Modulation of Nudt21 levels reveals dose-dependent roles of alternative polyadenylation in tissue regeneration

Domain: neuroscience
Published: 2026-01-24
Editorial outcome: minor_revision

Abstract
Stem cells continually self-renew and differentiate to sustain tissue homeostasis, yet the role of post-transcriptional mechanisms in guiding these processes remains incompletely understood. Here, we demonstrate that the regulation of 3’UTR length via alternative mRNA polyadenylation (APA) is essential for stem cell function across diverse tissues. Modulating the APA regulator Nudt21 reveals that stem cell self-renewal and differentiation depend on distinct dosage thresholds and thus can be uncoupled. Specifically, moderate Nudt21 suppression elicits a maturation arrest of stem cells due to 3’UTR-shortening of differentiation-associated mRNAs that escape miRNA regulation and perturb ceRNA networks. By contrast, complete Nudt21 suppression additionally shortens the 3’UTRs of mRNAs encoding essential multiprotein complexes, including the nuclear pore, leading to complex destabilization, proteotoxic stress, DNA damage, and cell cycle arrest. Critically, deletion of the alternative 3’UTRs of individual nucleoporins recapitulates defects observed with Nudt21 loss. We further demonstrate that the co-translational assembly of dozens of protein complexes is impaired in Nudt21-deficient cells, providing a mechanistic framework for compromised complex integrity. Collectively, our results show that APA plays distinct, dose-dependent roles in stem cell homeostasis by fine-tuning the expression of differentiation-associated genes and coordinating the biogenesis of multiprotein complexes essential for cell cycle progression.

Introduction
The maintenance of regenerative tissues typically relies on dedicated stem cells that continually self-renew and differentiate to replenish specialized cells lost during homeostasis or injury1. While the self-renewal and differentiation of tissue stem cells are known to be regulated by transcriptional and epigenetic mechanisms, the role of post-transcriptional mechanisms in stem cell regulation remains largely unexplored. Notably, over 50% of mammalian genes contain more than one polyadenylation signal (PAS), allowing mammals to greatly diversify the production of mRNA isoforms via APA2. APA has emerged as a crucial post-transcriptional layer of gene regulation that largely acts in parallel with conventional transcriptional mechanisms of gene expression. Changes in APA typically cause a lengthening or shortening of 3’UTRs, which facilitates the respective inclusion or exclusion of sequences targeted by miRNAs and RNA-binding proteins. This, in turn, can alter the stability and/or translation efficiency of affected transcripts3–9. Morerecently, APA has also been implicated in the subcellular localization of certain proteins, particularly surface molecules, as well as in the assembly of alternative cancer-associated protein complexes7,10,11. In addition to generating mRNA isoforms that differ exclusively in their 3’UTR (3’UTR APA), APA within introns (intronic APA) can give rise to mRNA isoforms that differ in their coding sequence due to the selection of alternative terminal exons12. Thus, 3’UTR APA and intronic APA have the potential to profoundly impact the function of proteins.

The cleavage and polyadenylation (CPA) machinery, which regulates the 3’ end processing of mRNAs, is composed of multiple subcomplexes with dedicated functions, including CPSF, CstF, CFIm, and CFIIm13–15. Of these subcomplexes, only CFIm (cleavage factor I) is dispensable for general mRNA 3’ end processing, but essential for directing 3’UTR APA16,17. Indeed, as many as 1,000 transcripts undergo 3’UTR changes upon siRNA-mediated knockdown of the CFIm components Nudt21 (also known as Cpsf5) or Cpsf618,19, which results in the destabilization of the entire CFIm complex19–21. In the absence of either Nudt21 or Cpsf6, the proximal PAS is favored to generate mRNA isoforms with short 3’UTRs, consistent with the CFIm complex functioning as an activator of distal PAS usage22. Accordingly, distal PAS sites are enriched for the Nudt21 binding motif UGUA compared to proximal PAS sites23. However, only a fraction of mRNAs undergoing 3’UTR shortening in the absence of Nudt21 produce increased protein levels, pointing to more complex roles of APA in gene regulation5,24. Together, these data indicate that the 3’ end processing of all mRNAs is controlled by the CPSF/CstF/CFIIm subcomplexes of the CPA machinery, while the APA of mRNAs with multiple PAS sites is additionally controlled by the CFIm complex.

Suppression of CFIm components, particularly Nudt21, has previously been reported to promote6 or suppress25,26 tumorigenesis in diverse cancer contexts. Moreover, the reduction of Nudt21 levels has been suggested to compromise preimplantation development27, certain neurological functions20,28,29 and pulmonary fibrosis30,31. Our lab previously showed that suppression of Nudt21 enhances the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) by upregulating critical chromatin factors that facilitate cellular plasticity18. Moreover, Nudt21 has been suggested to guide the activation of quiescent hematopoietic stem cells (HSCs) by controlling the switch between two different glutaminase 3’UTR isoforms32, and it reportedly modulates the expression of the B cell surface antigen CD19 in acute lymphoblastic leukemia33. While these studies implicate APA in the modulation of specific genes in particular experimental or disease contexts, its broader physiological roles and associated mechanisms remain largely unexplored.

Here, we investigate the functional significance of APA through conditional deletion of the key CFIm component Nudt21 in mice. Our findings reveal a previously unrecognized, essential role for APA in maintaining actively regenerating tissues, including various epithelia and the hematopoietic system, by ensuring the persistence of proliferative stem and progenitor cell populations. Through mechanistic dissection of Nudt21-deficient stem cells, we uncover a dose-dependent function of APA that (1) ensures lineage-specific gene expression important for differentiation, and (2) coordinates the stability of multiprotein complexes crucial for cell division in the context of stem cell self-renewal and progenitor cell proliferation. We highlight nucleoporins as central Nudt21 targets, whose APA safeguards the stability and function of the nuclear pore complex. Together, our results reveal that APA is indispensable for organismal survival by maintaining tissue homeostasis and suggest an APA-dependent mechanism by which essential multiprotein complexes are coordinately regulated in dividing cells to safeguard stem cell self-renewal and progenitor cell proliferation.

Results
CFIm is enriched in stem and progenitor cells of highly regenerative tissues
To determine whether Nudt21, a key component of the CFIm complex, is dynamically regulated during tissue homeostasis, we performed quantitative immunofluorescence (IF) analysis across three highly regenerative tissues containing defined stem, progenitor, and differentiated cell populations. We first examined the esophagus, which is a simple stratified squamous epithelium comprised of basal stem cells, which continuously self-renew and differentiate into suprabasal and superficial cells. While Nudt21 was detectable across the entire epithelium, we observed a gradient of Nudt21 levels that was highest, albeit variable, in Keratin (Krt) 14+ basal stem cells, lower in Krt14- suprabasal cells and lowest in the superficial layer of the epithelium containing the most differentiated cells (Fig. 1a). We similarly detected differential enrichment of Nudt21 levels in the intestinal epithelium, which is organized into glandular units containing highly proliferative stem and progenitor cellswithin the crypt compartment, as well as differentiated progeny within the villus compartment. Mirroring our observations in the esophagus, Nudt21 levels were highest in Lgr5+ intestinal stem cells and lowest in mature villus cells, with Lgr5- crypt-based progenitors exhibiting intermediate levels of Nudt21 (Fig. 1b). To explore whether the differential expression of Nudt21 within regenerative epithelia extends to a non-epithelial tissue with high turnover, we measured Nudt21 levels in the hematopoietic system. Indeed, Nudt21 was most abundant in HSCs and multipotent progenitors (MPPs) but less abundant in more committed myeloid progenitors (MyPs) and lineage-positive (Lin+) cells (Fig. 1c). We confirmed the differential expression of the CFIm components Nudt21, Cpsf6, and Cpsf7 across stem/progenitor and mature cell populations using published single-cell RNA-sequencing data for the intestine34 and esophagus35 (Supplementary Fig. 1a-d), as well as our own unpublished data of hematopoietic stem and progenitor cells (HSPCs) (Supplementary Fig. 1e and 1f). NUDT21 mRNA levels were also higher in human HSPCs compared to mature blood cell types using the bloodspot database36 (Supplementary Fig. 2a). Together, these observations indicate that CFIm expression is dynamically regulated in stem and progenitor cells across diverse tissues and species.

CFIm is required for the maintenance of diverse regenerative tissues
To probe the functional role of the CFIm complex in vivo, we generated Nudt21 knockout (Nudt21 KO) murine embryonic stem cells (ESCs) in which the RNA-binding domain essential for Nudt21 function has been flanked by loxP (fl) sites. These ESCs were then used to derive conditional Nudt21 (Nudt21fl/fl) mice (Fig. 1d). We crossed Nudt21fl/fl mice to Rosa26 (R26)-CreER mice, allowing us to delete Nudt21 ubiquitously following administration of tamoxifen. We tested the deletion of the targeted allele by exposing tail keratinocytes explanted from these animals to the tamoxifen analog 4-Hydroxytamoxifen (4OHT), which resulted in a loss of Nudt21 protein, as expected (Fig. 1e). In addition, we crossed Nudt21fl/fl mice to Keratin 5 (Krt5) CreER mice (Fig. 1d) to delete Nudt21 specifically in the basal stem cells of squamous epithelia including the esophagus where we observed elevated Nudt21 levels (Fig. 1a). Strikingly, the ubiquitous, acute loss of Nudt21 with R26-CreER led to the rapid morbidity of animals within 5-7 days. Mice with acute deletion of Nudt21 exclusively in Krt5+ squamous epithelia survived slightly longer and succumbed within 7-14 days (Fig. 1f). Together, these findings point to an essential role of the CFIm complex in the survival of adult mice.

To characterize possible phenotypes underlying the morbidity of Nudt21-deficient mice, we performed histology and marker analyses. Overall, ubiquitous Nudt21 deletion did not result in obvious defects in static tissues such as the kidney, heart and skeletal muscle (Supplementary Fig. 2b). However, when we examined animals with squamous tissue-specific Nudt21 loss, we observed a significant reduction in basal cell density in the esophagus (Fig. 1g top and 1h left), tongue and oral epithelium (Supplementary Fig. 2c), as well as morphologically abnormal cells within the suprabasal layers of these epithelia, suggesting a defect in basal stem cell self-renewal and differentiation. Consistent with this observation, we detected fewer Bromodeoxyuridine-positive (BrdU+) proliferative cells in the basal layer of the esophagus in these animals (Fig. 1g bottom and 1h right). Examination of actively regenerating tissues in ubiquitous Nudt21 KO mice further revealed a defect in the intestinal epithelium over time. Specifically, we observed a reduction in villus length and a decrease in BrdU+ cells in the crypt compartment, leading to the disintegration of the epithelium within 8 days (Figs. 1i and 1j, and Supplementary Fig. 2d). These results suggest a requirement for CFIm activity to sustain stem and progenitor cell populations across diverse epithelial tissues crucial for organismal survival.

We also noted that tamoxifen-induced ubiquitous Nudt21 KO mice exhibited reduced bone marrow cellularity, suggesting a role for CFIm in the hematopoietic system (Fig. 1k). To assess whether CFIm is required for hematopoiesis, we transplanted the bone marrow of untreated Nudt21fl/fl; R26-CreER (experimental) and Nudt21fl/fl (control) mice into irradiated recipient mice and then treated these with tamoxifen (Fig. 1l). While control mice had normal numbers of blood cells two weeks after tamoxifen treatment, most experimental mice became morbid and developed a severe pancytopenia as reflected by the decline in erythrocytes, platelets, and leukocytes in the peripheral blood (Fig. 1m). To confirm the cell autonomy of this phenotype, we generated mixed bone marrow chimeras containing either Nudt21fl/fl; R26-CreER or Nudt21fl/fl bone marrow in combination with wild-type bone marrow. Nudt21 deletion in these chimeras led to a selective loss of Nudt21-deficient HSCs, MPPs and mature blood cells without disrupting wild-type hematopoietic cells in the same animals, confirming an essential and cell-intrinsic requirement for Nudt21 in hematopoiesis (Supplementary Fig. 2e-2h). Collectively, our results establish that CFIm and thus APA are cell-autonomously required for the survival of adult animals by controlling the maintenance of stem and progenitor cell populations in various actively regenerating tissues.

CFIm maintains stem and progenitor cells by enabling cell cycle progression
Considering the profound effect Nudt21 loss has on stem and progenitor cells within actively regenerating tissues, we hypothesized that CFIm may be required for cell cycle progression. To this end, we established basal stem cell cultures from the esophageal epithelium of Nudt21fl/fl; R26-CreER mice and measured their distribution in the cell cycle phases, G1, S, and G2/M over time in the presence and absence of 4OHT (Figs. 2a, 2b). Compared to control cells, esophageal stem cells treated with 4OHT exhibited a progressive loss of S phase cells and a concomitant increase in G1 phase and G2/M phase cells (Figs. 2b-2d). By day 6 of 4OHT treatment, the majority of stem cells ceased to synthesize DNA and arrested in either G1 or G2/M. Acute Nudt21 deletion did not lead to increased apoptosis within the examined time window (6 days), although cells began to die shortly thereafter, indicating that cell death is a consequence rather than a driver of cellular defects incurred by the loss of CFIm function (Fig. 2e).

To examine whether Nudt21 loss disrupts cell cycle progression in other actively regenerating tissues, we explanted intestinal, hematopoietic, and skin cells from Nudt21fl/fl; R26-CreER mice in conditions that support the propagation of resident stem and progenitor cells (Supplementary Fig. 3a-3i). Paralleling our observations in esophageal cultures, intestinal, hematopoietic, and skin keratinocyte cultures exhibited a pronounced reduction of cells in S phase with an accumulation of cells at the G1 and/or G2/M phases of the cell cycle within 6 days of 4OHT treatment. These findings suggest that a CFIm-dependent cell cycle arrest of self-renewing stem cells and proliferative progenitors underlies the observed regenerative defects in animals.

CFIm is dispensable in quiescent stem cells unless they enter the cell cycle
As CFIm loss-of-function in stem cells of actively regenerating tissues elicits a profound self-renewal defect, we next asked whether CFIm is required for the maintenance of stem cells that have transiently exited the cell cycle. Skeletal muscle contains a rare population of stem cells termed satellite cells, which are quiescent in homeostasis but activated upon injury or explantation ex vivo. Once activated, satellite cells enter the cell cycle to proliferate and regenerate the injured muscle37–39. Analysis of published single-cell RNA-sequencing data40 revealed that Nudt21 is highly expressed in quiescent and activated satellite cells but downregulated in mature muscle cells (Supplementary Fig. 4a and 4b), consistent with our observations in epithelia and the bone marrow. To study CFIm function in quiescent satellite cells, we crossed Nudt21fl/fl mice to Pax7-CreER(gaka) mice41, which enable satellite cell-specific induction of Cre activity while maintaining endogenous Pax7 expression (Fig. 2f). We observed normal numbers of satellite cells and muscle mass in Nudt21fl/fl; Pax7-CreER mice at 30-40 days after tamoxifen treatment despite efficient Nudt21 deletion (Figs. 2g-2i), and these animals showed no overt muscle pathologies during this experimental time frame. These data suggest that CFIm function is dispensable for satellite cell maintenance in uninjured muscle. By contrast, we detected a severely impaired regenerative response upon cardiotoxin-mediated muscle injury of tamoxifen-treated Pax7-CreER (gaka); Nudt21fl/fl mice (Figs. 2j-2l), with reduced numbers of newly regenerated (centrally nucleated) myofibers and increased scar tissue deposition compared to controls. Consistent with a proliferation-dependent stem cell defect, satellite cells explanted from Nudt21fl/fl; Pax7-CreER mice failed to expand in culture, in contrast to satellite cells explanted from control animals (Figs. 2m-2o). Collectively, our results suggest that CFIm and hence APA are dispensable in quiescent muscle stem cells, but essential in activated muscle stem cells forced to proliferate in response to muscle damage in vivo or external stimuli ex vivo.

Modulation of CFIm levels uncouples self-renewal from differentiation
Previous studies by our lab and others have demonstrated that transient siRNA-mediated suppression of CFIm enhances the reprogramming of fibroblasts into iPSCs, and promotes tumor cell growth6,18,42. However, our findings here indicate that complete loss of CFIm function compromises stem cell self-renewal and drives subsequent failures of tissue regeneration, which are phenotypes that were not observed with siRNA mediated silencing. These differences imply that varying levels of CFIm, as observed in Nudt21 knockdown versus knockout approaches, may have distinct effects on cellular functions.

To test this hypothesis, we assessed the cellular and molecular consequences of reducing Nudt21 levels to different extents. To this end, we introduced a doxycycline (dox)-inducible hemagglutinin-tagged Nudt21 (HA-Nudt21) lentiviral transgene into Nudt21fl/fl; R26-CreER esophageal stem cells, allowing us to titrate Nudt21 levels in a Nudt21-deficient background (Fig. 3a). We then determined the effects of different Nudt21 levels on the self-renewal and differentiation potentials of esophageal stem cells. Reassuringly, dox titration in transduced esophageal cultures led to dose-dependent expression of Nudt21 protein (Fig. 3b bottom). Moreover, the expression levels of Cpsf6, another integral subunit of the CFIm complex, scaled with the expression levels of Nudt21 in our titratable system, consistent with Nudt21 being an essential and limiting component of the CFIm complex19–21. Leveraging this system, we determined that a reduction of Nudt21 levels down to ~25% of levels in control cells using 50 ng/ml dox is sufficient to support stem cell self-renewal and cell cycle progression (Fig. 3b top) for at least 8 passages. We refer to this condition as Nudt21 hypomorph. However, when Nudt21 levels dropped below ~25%, stem cells could not be maintained due to an irreversible cell cycle arrest.

To assess the impact of different Nudt21 levels on differentiation, we treated esophageal stem cell cultures with calcium, which triggers a rapid cell cycle exit and subsequent epithelial differentiation43 (Fig. 3c, Supplementary Fig. 4c and d). Specifically, we compared Nudt21 control cultures, Nudt21 KO cultures and Nudt21 hypomorph cultures. We determined that the calcium-induced cell cycle arrest occurs prior to the Nudt21 KO-induced cell cycle arrest, allowing us to assess whether Nudt21 suppression affects differentiation independently from the self-renewal defect noted above. Expression of the differentiation marker Keratin 13 (Krt13) was upregulated in control cells exposed to calcium, as expected. By contrast, Nudt21 hypomorph and KO cells failed to upregulate Krt13, suggestive of a differentiation block (Fig. 3c top and Supplementary Fig. 4d). Consistent with this observation, expression of the basal stem cell marker Trp63 was downregulated in control cells but aberrantly maintained in Nudt21 hypomorph and KO cells. The behavior of these two markers extended to other proteins, as differentiation-associated proteins were less effectively induced, while stem cell-associated proteins were retained in Nudt21 hypomorph and KO cells compared to control (Fig. 3c bottom). Critically, conditional KO mice acutely depleted for Nudt21 showed a similar reduction in Krt13 expression in primary esophagus tissue, demonstrating that our observations in cultured stem cells extend to an in vivo setting (Supplementary Fig. 4e). Together, these titration experiments indicate that Nudt21/CFIm act in a dose-dependent manner, thus uncoupling the differentiation defect from the self-renewal defect in Nudt21-depleted epithelial stem cells.

CFIm regulates polyA site usage in a dose-dependent manner
To understand molecularly how different Nudt21 levels exert distinct phenotypes in stem cells, we performed polyA site-sequencing44 (PAS-Seq) using Nudt21 control, KO and hypomorph esophageal stem cell cultures. Of all detected mRNAs (n=4,398), 52% or 2,272 mRNAs changed in 3’UTR length (i.e., shortening or lengthening) upon acute deletion of Nudt21 (Fig. 3d). Most of these changes involved a shortening of 3’UTRs from distal to proximal PAS sites (n=2,173 mRNAs), whereas only a minority of changes involved a lengthening of 3’UTRs from proximal to distal PAS sites (n=99 mRNAs), consistent with previous observations using Nudt21 knockdown strategies18,45. However, the number of mRNAs undergoing APA in Nudt21 KO cells was two to three-fold higher than in previous knockdown studies6,18, supporting the dose-dependent effects of Nudt21 depletion on APA patterns. The extent of APA changes in our hypomorph cells was significantly smaller compared to KO cells and resembled the scope of APA changes previously seen in Nudt21 knockdown experiments, with 955 mRNAs undergoing 3’UTR shortening and 41 mRNAs undergoing 3’UTR lengthening (Fig. 3e). Of note, most transcripts undergoing 3’UTR shortening in hypomorph cells (n=944 genes or 99%) overlapped with transcripts undergoing 3’UTR shortening in Nudt21 KO cells. By contrast, there was minimal overlap between transcripts undergoing 3’UTR lengthening in Nudt21 KO and hypomorph cells (n=27 genes or 24%), suggesting shortened mRNAs are common, direct targets while lengthened mRNAs are indirect targets of Nudt21 (Fig. 3f). Together, these observations reinforce our conclusion that Nudt21/CFIm acts as a dose-dependent regulator of APA and provide an experimental basis for defining relevant mediators of self-renewal and differentiation.

APA drives differentiation by modulating miRNA access to 3’UTRs
To narrow down CFIm targets underlying the differentiation block, we determined common APA changes between Nudt21 KO and hypomorph cells, considering that both genotypes lead to a marked differentiation block (Fig. 3c). We focused on the 944 transcripts commonly shortened in Nudt21 KO and hypomorph cells (Fig. 3f). Changes in APA usage have been associated with the elimination of miRNA target sites in the 3’UTRs of associated genes, which can lead to increased transcript and protein levels3,4,6,42,46 and consequently altered differentiation18,47–49 (Fig. 3h). We thus compared miRNAs expressed in basal stem cells (Supplementary Fig. 4f) with the 944 transcripts undergoing 3’UTR shortening in both Nudt21 KO and hypomorph cells to define potential matches. Strikingly, as many as 75% of shortened 3’UTRs but only ~30% of unchanged 3’UTRs contained binding sites for expressed miRNAs (Fig. 3g), suggesting that the majority of Nudt21 targets undergoing shortening in both Nudt21 KO and hypomorph cells are likely regulated by miRNAs.

To pinpoint relevant candidates among the 944 commonly shortened transcripts that follow this mode of gene regulation, we integrated our RNA-Sequencing, miRNA-Sequencing and proteomics data from Nudt21 KO and hypomorph cells, arriving at 24 high-confidence targets with increased RNA and protein levels (Fig. 3i). For example, we detected binding sites for 11 expressed miRNAs within the 3’UTR of the histone demethylase Jmjd1c previously validated as a relevant Nudt21/APA target during iPSC reprogramming18. Similarly, we detected binding sites for 28 expressed miRNAs within the 3’UTR of the COPII vesicle component Sec24a. Nudt21 KO and hypomorph cells exhibited a shift from distal to proximal PAS usage in the 3’UTRs of Jmjd1c and Sec24a, which is expected to exclude binding sites for cognate miRNAs (Fig. 3j)46,47.

To assess whether the aberrant upregulation of Jmjd1c or Sec24a contributes to the differentiation block of Nudt21-depleted cells, we suppressed each gene using siRNAs in Nudt21 hypomorph esophageal stem cells undergoing differentiation. Remarkably, suppression of either Jmjd1c or Sec24a expression ameliorated the differentiation block, as determined by upregulation of the differentiation marker Krt13, and downregulation of the basal cell marker Trp63 (Figs. 3k and 3l). Of note, the long 3’UTR isoforms of Jmjd1c and Sec24a were detectable throughout differentiation in control cells, implying that miRNAs are continuously required to suppress these targets (Supplementary Fig. 4g). To determine whether specific miRNAs targeting Jmjd1c and Sec24a 3’UTRs underlie the differentiation defect of Nudt21 hypomorph cells, we silenced miR-449a-5p, miR-30d-5p, and miR-34a-5p using anti-miRs in wild-type esophageal cells. The silencing of miR-34a, which targets Jmjd1c, delayed differentiation akin to Nudt21 hypomorphs, as measured by elevated Trp63 expression (Supplementary Fig. 4h). Notably, some anti-miRs decreased p63 levels, pointing to complex regulatory interactions between mRNAs and their cognate miRNAs. Collectively, these findings illustrate that the differentiation block in Nudt21-deficient cells is driven, at least in part, by the dysregulated post-transcriptional control of key miRNA targets including Jmjd1c and Sec24a.

APA rewiring impacts global ceRNA networks
In addition to mediating individual mRNA-miRNA interactions, 3’UTRs have previously been implicated in the co-regulation of mRNAs with shared miRNA binding sites. As these co-regulated mRNAs typically compete for the same miRNAs, they are often referred to as competing endogenous RNAs (ceRNAs)50. To test whether widespread 3’UTR shortening via Nudt21 modulation influences ceRNA networks, we grouped mRNAs based on common miRNA binding sites using wild-type esophageal stem cells. RNA pairs were defined as ceRNAs if they were co-regulated across biological replicates (>0.6 Pearson’s correlation coefficient). In wild-type cells, 41.9% of mRNA pairs behaved as ceRNAs based on these criteria (Supplementary Fig. 5a). However, in Nudt21 KO cells, only 27.7% of mRNA pairs were classified as ceRNAs (Supplementary Fig. 5b), while in Nudt21 hypomorph cells, 36.3% of mRNA pairs qualified as ceRNAs (Supplementary Fig. 5c). Inspection of mRNAs co-regulated in this fashion revealed the aforementioned Sec24a transcript as a bona fide ceRNA. Sec24 shows strong co-regulation with dozens of other mRNAs targeted by the same miRNAs including the chromatin regulators Dnmt3a and Baz2b (Supplementary Fig. 5d). However, this co-regulation is severely disrupted in Nudt21 KO and hypomorph cells (Supplementary Fig. 5e and f). Thus, loss of the long 3’UTR isoform of Sec24a appears to disrupt a strong ceRNA network by altering the corresponding miRNA target landscape. More generally, these observations reinforce our conclusion that APA modulates miRNA-dependent gene expression and associated ceRNA networks critical for differentiation42.

APA sustains stem cell self-renewal by maintaining essential multiprotein complexes
To define APA targets underlying the self-renewal defect of Nudt21-deficient cells, we performed multiplexed mass spectrometry-based proteomics51 of control and Nudt21 KO cells at different time points after 4OHT treatment (0, 2, 4, 6 days), as well as Nudt21 hypomorph cells at day 6 (Hd6). As endogenous Nudt21 protein levels were fully depleted only by day 4 of 4OHT treatment (Fig. 4a), we determined differentially expressed proteins (DEPs) that were commonly deregulated on days 4 and 6 (n=1,677) in Nudt21 KO cells (Fig. 4b). Moreover, since Nudt21 hypomorph cultures self-renew normally, we focused only on those 353 candidates that changed protein levels and 3’UTR length in Nudt21 KO cells but not in hypomorph cells. Given Nudt21’s crucial role in self-renewal, we selected essential genes from the remaining 353 candidates based on the publicly available ‘essentialome’ dataset DepMap52, leading to 73 essential candidates for final analysis (Fig. 4b). Of note, the majority of these candidate proteins (55 out of 73 proteins (75%) at day 4, and 58 out of 73 proteins (80%) at day 6) were downregulated in the Nudt21 KO cells (Fig. 4c), suggesting a strong disruption of essential cellular proteins (Fig. 4b). Indeed, when we examined the expression levels of all essential genes detected in our proteomics dataset (n=4,386) regardless of whether they are Nudt21 targets, we observed a progressive downregulation of these genes in Nudt21 KO cells starting on day 4, but stable expression in Nudt21 hypomorph cells (Supplementary Fig. 6a). These results imply that Nudt21 deficiency impacts diverse essential cellular processes beyond direct Nudt21 targets. Conversely, non-essential genes (n=21,768) exhibited a subtle yet significant upregulation in Nudt21 KO and hypomorph cells (Supplementary Fig. 6b), consistent with an escape from miRNA-mediated suppression (see Fig. 3). In line with this interpretation, non-essential genes were more likely to be targeted by miRNAs than essential genes (Supplementary Fig. 6c). Consistent with their essentiality, the 73 candidate genes have more PAS sites than expected by chance, and this enrichment is conserved between mouse and human, pointing to common regulatory roles of associated 3′UTRs (Supplementary Fig. 6d and e). Together, these data suggest that APA plays distinct and opposing roles in the regulation of proteins driving self-renewal (downregulated with Nudt21 loss) and differentiation (upregulated with Nudt21 loss via miRNAs).

To gain insights into the regulation of the 73 candidates associated with the self-renewal defect of Nudt21 KO stem cells, we compared the dynamics of their RNA and protein levels using our RNA-Sequencing and proteomics data, respectively (Fig. 4d). We focused on day 4 after Nudt21 deletion since cells have not yet undergone cell cycle arrest (Fig. 2b), yet endogenous Nudt21 levels are fully depleted (Fig. 4a) and the global 3’UTR landscape has been rewired (Fig. 3d and e). Further emphasizing the significance of day 4 as a critical time point, we found that a dox-inducible Nudt21 transgene could rescue the cell cycle arrest of conditional Nudt21 KO cells when dox was added on day 1, 2, or 3, but not when added on day 4 after Nudt21 deletion (Fig. 4e and f). This result indicates that day 4 Nudt21 KO cells have embarked on an irreversible path towards cell cycle arrest, which manifests at day 6. Importantly, protein levels for the 73 essential candidate genes decreased at day 4 even though associated RNA levels remained unchanged in Nudt21 KO cells (Fig. 4d). Hypomorph cells largely retained RNA and protein expression of these candidates, as expected. We further showed that protein downregulation in Nudt21 KO cells was not due to an impaired translational efficiency of associated RNAs using polysome sequencing (Fig. 4d). These results indicate that acute disruption of the CFIm complex elicits downregulation of candidate proteins through 3’UTR shortening of corresponding mRNAs, but without altering mRNA levels or translational efficiency. Gene Ontology (GO) analysis of the 73 deregulated proteins revealed several protein subunits of the nuclear pore (e.g., Nup160, Seh1l, Nup107), the mitochondrial ribosome (e.g., Mrpl33, Mrpl 35, Mrpl37), the nucleolus (e.g., Ddx54, Dhx33, Nifk) and the spliceosome (e.g., Alyref, Srsf1, Hnrnpm) among the downregulated proteins (Fig. 4d right). These results suggest that multiprotein complexes are uniquely sensitive to destabilization upon complete loss of Nudt21 function. By contrast, GO analysis of upregulated proteins revealed pathways associated with “Regulation of protein-containing complex assembly”, “Autophagy” and “Protein folding”, consistent with defects in protein homeostasis (Supplementary Fig. 7a). Indeed, chaperones involved in “protein folding” were strongly upregulated at the protein level in Nudt21 KO cells, consistent with the activation of a cellular stress response caused by imbalanced protein levels, rather than APA (Supplementary Fig. 7b-d).

As the integrated stress response (ISR) pathway is typically activated in contexts of proteotoxic stress, we treated Nudt21 KO cells with the ISR inhibitor ISRIB53–55. This led to a subtle yet significant increase in proliferation, suggesting that proteotoxic stress indeed contributes to the observed cell cycle defects of Nudt21 depleted cells (Supplementary Fig. 7e).

To confirm and expand this analysis with an orthogonal approach, we queried our unfiltered DEPs using the EBI Complex Portal database56, which encompasses all annotated protein complexes, and then calculated enrichment scores for each complex in Nudt21 KO cells compared to wild-type controls (Fig. 4g). Strikingly, proteins downregulated in Nudt21 KO cells were assigned to 21 different protein complexes, whereas upregulated proteins were assigned to only 6 different protein complexes, reinforcing the notion that CFIm disruption leads to the preferential downregulation of multiprotein complexes. Top-scoring downregulated complexes included again the 39S mitochondrial large ribosomal subunit, the 40S cytosolic small ribosomal subunit and the nuclear pore, as well as several histone acetyltransferase complexes (TFTC, NSL, NuA4) and the telomerase holoenzyme. Of these, the nuclear pore stood out in that it contained the most subunits (40%) with APA changes compared to the other two top-scoring ribosomal complexes (9% each) in Nudt21 KO cells. Moreover, the integrity of the nuclear pore is critical for the assembly of ribosomes and could thus function upstream of the other top-scoring complexes57. The observed disruption of entire protein complexes despite the 3’UTR shortening of only some of its subunits in Nudt21 KO cells is consistent with cellular protein quality mechanisms, whereby the elimination of individual subunits elicits the proteasome-dependent degradation of remaining, unpaired orphan subunits58. Together, these observations indicate that essential APA targets such as nucleoporins are destabilized specifically in Nudt21 KO cells while they remain intact in Nudt21 hypomorph cells, providing a plausible mechanism for the observed self-renewal defect of Nudt21 KO stem cells.

CFIm stabilizes nuclear pore to facilitate mRNA export and genomic stability
Focusing on the nuclear pore complex, we observed that in total, 14 nucleoporins exhibited 3’UTR shortening with Nudt21 loss, which accounts for almost half of all nucleoporins (n=30) (Figs. 5a and 5b). A disproportionate number of these 14 nucleoporins belong to the so-called outer ring structure (e.g., Ahctf1, Nup160, Seh1l, Nup107, and Ndc1) required for nuclear pore stability and mRNA export59. Associated proteins were downregulated upon Nudt21 loss, suggesting a major nuclear pore defect (Fig. 5c). To determine whether nuclear pores are dysfunctional in Nudt21-depleted cells, we stained Nudt21 KO, hypomorph and control esophageal stem cell cultures with the nuclear pore antibody mAb414. Indeed, Nudt21 KO cells showed a severe diminution of nuclear pore signal (Figs. 5d and 5e), while hypomorph cells retained control-like signal (Supplementary Fig. 8a and 8b). Supporting a functional defect in nuclear pores, we also detected a marked decrease in cytoplasmic mRNA signal in Nudt21 KO cells using RNA fluorescence in situ hybridization (FISH) analysis, suggesting disrupted APA leads to a pronounced nucleocytoplasmic mRNA export defect (Figs. 5f and 5g). To rule out the possibility that Nudt21 directly interacts with and stabilizes nuclear pores, we performed co-IP/MS for HA-Nudt21 in self-renewing Nudt21 hypomorph cells (Supplementary Fig. 8c). Although we captured the entire CFIm complex including Nudt21, Cpsf6, and Cpsf7, as expected, we detected no nucleoporins in association with Nudt21, suggesting that the RNA-binding function of Nudt21 rather than physical interactions with nucleoporins underlie the defect in nuclear pore stability in Nudt21 KO cells.

If nucleoporins are relevant targets of the CFIm complex, we would expect their depletion to phenocopy aspects of Nudt21 loss. We therefore suppressed the nucleoporins Nup107, Nup160 and Seh1l using siRNAs in esophageal stem cells and measured effects on cell cycle progression (Supplementary Fig. 8d-8f). Indeed, suppression of Nup160 and to a lesser extent that of Nup107 and Seh1l led to an S phase arrest and a concomitant loss of nuclear pore staining in esophageal stem cells akin to Nudt21 KO cells. Of note, transient, near-complete suppression of Nudt21 with potent siRNAs also impaired Nup160 expression, but not Nup107 and Seh1l expression, suggesting that Nup160 stability is directly controlled by the CFIm complex. Moreover, Nudt21 deletion as well as nuclear pore depletion by siNup107 or siNup160 displayed an increased frequency of chromatin bridges, consistent with chromosomal instability, a known downstream consequence of nuclear envelope defects60,61 (Supplementary Fig. 8g and 8h), thus further strengthening the connection between APA and the nuclear pore. Together, these results uncover a previously unknown functional link between APA and the nuclear pore complex by placing specific nucleoporins downstream of CFIm.

Nucleoporin 3’UTRs are essential for nuclear pore stability and function
To assess whether the APA change of any particular nucleoporin(s) in Nudt21 KO cells drives the self-renewal arrest, we conducted a 3’UTR-focused CRISPR/Cas9 screen. Briefly, we lentivirally introduced a pool of 32 dual guide RNAs (dgRNA) targeting the distal (i.e., alternative) 3’UTRs of the 14 candidate nucleoporins undergoing APA in Nudt21 KO cells (see Fig. 5a) into wild-type esophageal stem cells containing a dox-inducible Cas9 transgene. Transduced cultures were propagated for 3 passages in the absence or presence of dox before quantifying dgRNA abundance by dgRNA sequencing (Fig. 5h). Depletion of specific dgRNAs indicates that associated 3’UTR elements are required for the self-renewal of esophageal stem cells akin to the Nudt21 KO phenotype. Interestingly, dgRNAs targeting the distal 3’UTRs of the outer ring nucleoporins Nup160 and Nup98/96 showed the strongest depletion, indicating these non-coding elements are required for continual self-renewal (Fig. 5i).

Considering that dgRNAs targeting Nup160 scored the highest in our self-renewal assay, we focused on this particular protein for the remainder of the study. Closer examination of our PAS-Seq data revealed that the Nup160 gene produces 3 different APA isoforms (Fig. 5j), which we designated Nup160 short (Nup160S), medium (Nup160M) and long (Nup160L). In asynchronously dividing esophageal stem cells, Nup160M was the dominant isoform while Nup160S and Nup160L were minor isoforms. To determine whether the low expression levels of Nup160L are due to cell cycle dependent effects, we purified esophageal basal cells at the G1, S and G2/M phases of the cell cycle and performed Nup160 isoform-specific qPCR analysis. Indeed, Nup160L levels transiently increased during the S and G2/M phases (Supplementary Fig. 9a and 9b), pointing to a cell cycle-dependent regulation of APA62. Of relevance, the dynamic expression of the Nup160L isoform coincides with the upregulation of nucleoporins and the assembly of nuclear pores during the late S and G2/M phases of the cell cycle (Supplementary Fig. 9c-9f), suggesting a possible regulatory role of Nup160L in nuclear pore homeostasis.

Nudt21 deletion led to a slight increase in Nup160S and a decrease in Nup160M expression but a complete loss of Nup160L expression, which was the isoform that scored in our pooled CRISPR/Cas9 screen (Fig. 5j). Accordingly, deletion of the distal-most 3’UTR exclusively associated with Nup160L with distinct dgRNA pairs led to a pronounced defect in S phase progression, nuclear pore signal and mRNA export (Figs. 5j-5p). By contrast, dgRNAs targeting the centrally located 3’UTR associated with Nup160M yielded cells with only a mild defect in cell cycle progression and nuclear pore signal intensity, suggesting this region is largely dispensable for cell proliferation (Supplementary Fig. 9g-9k). Similar to Nudt21 KO cells, deletion of the 3’UTR associated with the Nup160L isoform also resulted in the downregulation of the nuclear pore outer ring proteins Nup160, Nup107, and Seh1L, consistent with a destabilization of the entire outer ring structure (Supplementary Fig. 9l). We confirmed the crucial role of the Nup160L isoform in maintaining cell cycle progression and nuclear pore integrity using isoform-specific siRNAs (Supplementary Fig. 9m-9r). Interestingly, depletion of the Nup160L isoform via either CRISPR or siRNA approaches was also sufficient to elicit chromatin bridges typically observed in Nudt21 KO cells (Figs. 5q and 5r). However, deletion of the Nup160M 3’UTR did not cause chromatin bridges, underscoring the specific role of the long isoform in maintaining the self-renewal of esophageal stem cells by preserving nuclear and genomic integrity61,10. Notably, loss of Nudt21 in quiescent satellite cells did not diminish nuclear pore signal intensity, suggesting that dividing cells are uniquely sensitive to nuclear pore loss upon Nudt21 KO (Supplementary Fig. 9s and 9t). Together, these results establish that the outer ring nucleoporin Nup160 is a direct and crucial target of the CFIm complex responsible for maintaining nuclear pore integrity and consequently genomic integrity and stem/progenitor cell maintenance via regulation of its 3’UTR.

Finally, we tested whether the re-expression of specific Nup160 mRNA isoforms is sufficient to rescue the cell cycle defect of Nudt21 KO cells. Briefly, we introduced in vitro transcribed (IVT) RNAs encoding Nup160S, Nup160M and Nup160L into wild-type and Nudt21 KO cells. None of these IVT isoforms was able to rescue the cell cycle phenotype, and exogenous Nup160L RNA even exacerbated the Nudt21 KO-dependent cell cycle defect. This finding implies that Nup160 isoform levels need to be tightly regulated in cells, with aberrant up- or downregulation disrupting Nup160 function in dividing cells (Supplementary Fig. 10a and 10b). We tested the role of these 3’UTR isoforms in protein localization by leveraging chimeric fusion transcripts between Nup160 or eGFP coding regions and the three Nup160 3’UTR variants (Supplementary Fig. 10c). Notably, eGFP-Nup160L mRNAs transfected into esophageal stem cells yielded lower eGFP signal compared to eGFP-Nup160S or eGFP-Nup160M mRNAs 24 hours post transfection, pointing to a possible role of these RNA isoforms in the translational control of Nup160 (Supplementary Fig. 10d-g). In agreement with this model, we discovered that a biotinylated Nup160L mRNA probe associates with ribosomal proteins more efficiently than biotinylated Nup160M and Nup160S mRNA probes (Supplementary Fig. 11a-c). Of note, transfection of cells with the eGFP-Nup160L mRNA isoform but not the eGFP-Nup160M or eGFP-Nup160S mRNA isoforms triggered the formation of ectopic peri-nuclear foci 2-3 hours after transfection, which is reminiscent of condensates previously associated with nuclear pore homeostasis63,64 and warrants further investigation (Supplementary Fig. 11d and 10e).

APA impacts co-translational protein complex assembly
The above-mentioned observations raised the possibility that global 3′ UTR shortening might affect the assembly of protein complexes in Nudt21 KO cells. Large protein complexes tend to assemble via co-translational mechanisms whereby an nascent polypeptide encoding for one subunit interacts with another nascent polypeptide (co-co-translational assembly) or with a fully folded polypeptide (co-post-translational assembly) encoding for a different subunit65,66. To determine whether disruption of APA patterns alters the co-co-translational mode of protein complex assembly, we performed Disome Selective Profiling (DiSP)65,66 of ribosomes using control and Nudt21 KO cells. Of 4,986 mRNAs actively translated in control cells, 143 mRNAs (3%) met the criteria for co-co-translational assembly, which is in line with previous observations using transformed cell lines65 (Fig. 6a). Strikingly, nearly 44% (n=63) of these interactions were reduced in Nudt21 KO cells and about half of the associated mRNAs were Nudt21 targets, suggesting APA plays a crucial role in their co-translational regulation (Fig. 6b and c). Supporting the aforementioned role of Nudt21 in cell cycle progression, we identified pivotal regulators of mitosis among the most significantly altered mRNAs (Fig. 6d), including the Nudt21 targets Cenpf and Smc5 involved in chromosome segregation (Fig. 6e). We note that Nup160 itself did not score in our DiSP assay, which is likely due to an alternative co-translational or post-translational mode of assembly67,68. Collectively, these results suggest that Nudt21-dependent APA dynamics coordinate mitotic progression and protein complex homeostasis, processes that are crucial for maintaining the proliferative state and regenerative potential of several adult tissues.

Methods
Inducible HA-Nudt21 lentiviral constructs
To construct an inducible HA-Nudt21 lentiviral vector, we synthesized an N-terminally tagged HA-Nudt21 gBlock (IDT) and cloned it into a BamHI and EcoRI-linearized pCW57 (#78933) vector using Gibson Assembly (NEB). This method allowed for the insertion of HA-Nudt21 downstream of the TRE promoter, resulting in the deletion of Turbo RFP. Lentiviral particles were produced as previously described93.

Mice
The following mouse strains were purchased from The Jackson Laboratory (jax.org): K5CreER (Jax 029155), Lgr5-EGFP-IRES-CreER (Jax 008875), Rosa26-CreER (Jax 008463), KH2-iCas9 (Jax 029415) Nudt21fl/fl (this study, see below). Conditional Nudt21 KO mice were generated by crossing Nudt21fl/fl with one of the following Cre allele bearing mice: Rosa26CreER (ubiquitous KO), K5CreER (squameous epithelia KO) and Pax7-CreER (satellite cell KO). Control mice were either Nudt21 wt/fl and the corresponding CreER or Nudt21fl/fl without any CreER. For experiments, we administered tamoxifen into adult mice between 3 and 6 months of age. Both male and female mice (approximately equal proportions) were used in all experiments. No sex-specific differences were observed or analyzed, and no sex-related effects were expected for the biological processes under investigation. Mice were housed in ventilated cages on a standard 12h:12h light cycle. All procedures involving mice adhered to the guidelines of the approved Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC) protocol no. 2006N000104.

To generate bone marrow chimeric mice through bone marrow transplantation, 6-8 week-old B6.SJL (CD45.1) recipient mice were irradiated with a total dose of 1,200 rad (600 rad twice, with a 3-hour interval). Bone marrow was harvested from uninduced donor mice (Nudt21fl/fl; Rosa26CreER (KO) or Nudt21fl/fl (control)). Each recipient received 2,000,000 bone marrow cells from the donor mouse, injected retro-orbitally in 100μl of FBS-free PBS. The mice were then housed for 10 weeks to ensure complete engraftment before tamoxifen injection). For competitive bone marrow transplantation, 6-8 week-old B6.SJL (CD45.1) recipient mice were irradiated with a total dose of 1,200 rad (600 rad twice, with a 3-hour interval). Bone marrow was harvested from uninduced donor mice (Nudt21fl/fl; Rosa26CreER or Nudt21fl/wt; Rosa26CreER, or Nudt21fl/fl) and a competitor mouse (B6.SJL (CD45.1)). Each recipient received 1,000,000 bone marrow cells from the donor mouse and 1,000,000 cells from the competitor mouse, or the specified number of HSCs from the donor mouse and 500,000 bone marrow cells from the competitor mouse. The cells were injected retro-orbitally in 100μl of PBS. The mice were then housed for 8 weeks to ensure complete engraftment. Control mice were either Nudt21 wt/fl with the corresponding CreER or Nudt21fl/fl without any CreER. Both KO and control groups were treated with tamoxifen in the same manner.

ES cell culture and Nudt21 gene targeting
Mouse embryonic stem cells (Nudt21fl/fl, HEPD0770_7_B01 from IMPC) were grown on irradiated mouse embryonic fibroblasts in a growth medium consisting of KO-DMEM (Life Technologies) with 15% fetal bovine serum (FBS) (Hyclone), 1X L-glutamine (Life Technologies), 100U/mL Penicillin, 100μg/mL Streptomycin (Life Technologies), 1X MEM Non-Essential Amino Acids Solution (Life Technologies), and 50μM β-mercaptoethanol (Life Technologies). For selection casette removal, cells were grown to 80% confluence, then trypsinized and collected. The cells were washed once in PBS and resuspended in 500μL of PBS. Subsequently, 40μg of pCAGGS-flpE-puro were added, and the mixture was transferred to a 4mm electroporation cuvette (Biorad). Electroporation was performed using a Gene Pulser Xcell system (Biorad) in time constant mode with the parameters set to voltage=800V and time constant=0.2ms. The cells were then plated at clonal density onto DR4 irradiated mouse embryonic fibroblasts (GlobalStem) in a 6cm dish and cultured in ES cell medium for 24 hours. Afterward, the medium was replaced with growth medium containing 200μg/mL hygromycin (Gibco) to select for targeted clones. Individual colonies were picked, expanded, and tested via PCR to confirm proper integration.

Blastocyst Injection and generation of conditional Nudt21 KO mice
To create transgenic mice, Nudt21fl/fl ES cells were injected into E3.5 blastocysts as described in previous studies52,53. The resulting high-grade chimeras from Nudt21fl/fl ES cells were bred with B6/C57 wild type mice, and the offspring were genotyped to confirm germline transmission. These mice were then crossed with mice carrying Rosa26CreER or K5-CreER on a B6/C57 background. Mice of both genders, aged 8-16 weeks, were used in the study and induced, unless otherwise specified, with 5 daily doses of 20 mg/ml tamoxifen in corn oil. The mice in this study were housed and bred in Specific Pathogen Free (SPF) rooms at the AAALAC-accredited Center for Comparative Medicine vivarium at Massachusetts General Hospital. They were kept in ventilated cages with a standard 12:12 light cycle. All procedures involving mice followed the guidelines of the approved Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC) protocol no. 2006N000104.

Tamoxifen treatment
For in vivo tamoxifen treatment, both KO and control mouse were treated with tamoxifen. Tamoxifen (20mg/ml, T5648-1G, Sigma-Aldrich) was prepared in corn oil (Sigma-Aldrich), and each mouse received a daily intraperitoneal injection of 100µl for five consecutive days. For the satellite cell experiments, the mice received the same five doses of tamoxifen and were subsequently maintained on a diet containing 500mg/kg tamoxifen (Envigo) for 30-40 days. For in vitro tamoxifen treatment, cells were derived from conditional Nudt21 KO or control mice and cultured under specific conditions (detailed below). The media contained either ethanol (ETOH) as a control or (Z)-4-Hydroxytamoxifen (4OHT, Sigma-Aldrich, H7904-5MG) to induce Nudt21 KO. The 4OHT stock solution was prepared by dissolving 4OHT powder in ETOH to a concentration of 5 mg/ml (13 mM). To test for Cre toxicity94, new 4OHT batches were titrated on Nudt21 wt/wt; Rosa26CreER esophageal basal cells, skin keratinocytes, intestinal organoids, and hematopoietic progenitors. The effect of 4OHT on the cell cycle was assessed by EdU staining and FACS. The maximum tolerable concentration of 4OHT without impacting the cell cycle was approximately 0.1-0.05µM for all cell types. This concentration was used throughout all in vitro experiments, with 4OHT added at each media change. The 4OHT stock solution remained stable for up to 3 months.

Histology
Mice or tissues were fixed in Bouin’s solution (Sigma-Aldrich) or 10% formalin solution (Sigma-Aldrich), respectively. The fixed tissues were then prepared for IF, immunohistochemistry and H&E staining.

Immunofluorescence (tissues)
Immunohistochemistry staining was performed by iHisto Inc. (ihisto.io) Samples were processed, embedded in paraffin, and sectioned at 4μm. Paraffin sections were then deparaffinized and hydrated using the following steps: 15 min in xylene twice; 5, 5, and 5 min in 100%, 100%, and 75% ethanol, respectively; and 5 min in PBS at RT repeated three times. Antigen retrieval was achieved by boiling the sections in Citrate-based unmasking buffer for 115 min in pressure cooker and 20 min of cooling at RT. Slides were washed and blocked with bovine serum albumin (BSA) at RT for 20 min. Following blocking, sections were incubated with the primary antibodies diluted in BSA overnight at 4°C. The following day, slides were washed as above. Secondary antibodies were applied to sections and allowed to incubate for 1 h at RT. Slides were washed as above. Slides were stained with Sudan Black for 15 min to quench background and autofluorescence. Slides were washed under running water for 15 min. Slides were washed three times for 5 min each in TBS, TBS, and TBST, respectively. Slides were counterstained with DAPI and coverslipped using Fluoroshield (SIGMA).
Primary antibodies: goat anti-GFP (abcam, ab6673) 1:500, Nudt21 (Santa Cruz, 2203C3) 1:50, rabbit anti-Krt14 1:1000 (biolegend, 905304). Secondary antibodies (all from Thermofisher): donkey anti-goat Alexa flour 488, Donkey anti-mouse AF555, Goat anti, mouse Alexa flour 488, Goat anti-rabbit AF555

Immunohistochemistry
Immunohistochemistry (IHC) staining was performed by iHisto Inc. (ihisto.io) Samples were processed, embedded in paraffin, and sectioned at 4μm. Paraffin sections were then deparaffinized and hydrated using the following steps: 15 min in xylene twice; 5, 5, and 5 min in 100%, 100%, and 75% ethanol, respectively; and 5 min in PBS at RT repeated three times. Antigen retrieval was achieved by boiling the sections in Citrate-based unmasking buffer for 115 min in pressure cooker and 20 min of cooling at RT. A peroxidase blocking solution was applied to sections and allowed to incubate for 10 min and 2.5% normal horse serum for 30 min. After 30 min, excess serum was removed from the sections and primary antibody diluted in 2.5% normal horse serum was applied. Sections were incubated with diluted primary antibody overnight at 4°C. Sections were incubated in secondary antibody for one hour at RT. Slides were washed as before. The DAB working solution was applied to sections and the stain was allowed to develop. Slides were counterstained with hematoxylin. Slides were dehydrated in an oven set to 37°C and coverslipped using a xylene-based mounting medium. Primary Antibodies: mouse anti-Brdu (GB12031), (Santa Cruz, 2203C3) 1:50.
Secondary antibody: HRP Horse Anti-Mouse IgG Polymer Kit (ImmPRESS), Peroxidase (MP-7402, Vector Laboratories).

H&E staining
H&E staining was performed by iHisto Inc. (ihisto.io). Samples were processed, embedded in paraffin, and sectioned at 4μm. Paraffin sections were then deparaffinized and hydrated using the following steps: 15 min in xylene twice; 5, 5, and 5 min in 100%, 100%, and 75% ethanol, respectively; and 5 min in PBS at RT repeated three times. After deparaffinization, 4‑µm‑sectioned samples were placed on glass slides and stained with hematoxylin for 3 min and Eosin for 5 min. The slides were dehydrated by graded alcohol and coverslipped using a xylene-based mounting medium.

Excision PCR
To quantify knockout efficiency, cells were treated as described in the results section and genomic DNA was isolated using the Monarch Genomic DNA Purification Kit (NEB). PCR amplification was performed with the GoTaq Master Mix (Promega) by combining 5 µM primers with 10–50 ng of genomic DNA. The primers used for the reactions were as follows:
Nudt21_1: 5’-ACCAGTGAGGTGGCTCTAAGG-3’
Nudt21_2: 5’-AGGTCCCTGAAATGCTTAGCC-3’
Nudt21_3: 5’-AGGATCCAAGTTTTCAGATATTCTCCCC-3’
Nup160_long_1: 5’-AAACTTACGGCTCTCCACTACAGCATAAAAGGC-3’
Nup160_long_2: 5’-GTACCTGTATGTATGTGGCGGCGAGAGG-3’
Nup160_med_1: 5’-ACAGAAACCCTGTCTCAAGGC-3’
Nup160_med_2: 5’-GATTGAGGGCCTTGGATCTGCT-3’
The PCR program consisted of an initial denaturation step at 98°C for 2 min, followed by 35 cycles of denaturation at 98°C for 15s, annealing at 58°C for 20s, and extension at 72°C for 5 min. A final extension was carried out at 72°C for 10 min. The PCR products were then electrophoresed alongside a 1kb DNA Ladder (NEB) on a 1% agarose gel.

2D epithelial cell culture
Long-term expansion of diverse epithelial basal cells was conducted as previously described95. Briefly, adult esophagi or tails were removed from mice, cleaned in ice-cold PBS, and rocked for no more than 10 h at 4°C in dissociation medium composed of DMEM/F12 (Gibco), 200 μg/ml Pronase (Sigma-Aldrich), 10 μg/ml DNase I (Worthington), 1% Fraction V BSA (Gibco), 1× pen/strep, and 10 μM Y-27632 (Tocris). The epithelium from the esophagi or tail was dissociated from the dermis using angled forceps (FST 00276-13). The epidermis was cut into ~2–5mm pieces, suspended in cold PBS, pipetted up and down, and passed through a 100 μm mesh placed on a 50 ml conical tube. The epithelium on the mesh was ground with a syringe plunger, and PBS was flushed through the mesh to recover epithelial cells. Dissociated epithelial cells were explanted onto T25 flasks pre-coated with conditioned medium collected from 804G cells. Complete SAGM medium, supplemented with 10 μM Y-27632 (Tocris), 1 μM CHIR99021 (Tocris), 1 μM A83-01, and 1 μM DMH-1, was used for expansion. Cells were kept in the exponential growth phase (maximum 8000 cells/cm²). Media changes were performed every 48h. Cells were dissociated from the flasks and dishes either by scraping or treatment with TrypLE (Gibco) for 10 min. Epithelial cells were collected in 1% BSA/PBS (Invitrogen) and spun down at 300×g. Cells were kept in culture for a maximum of 6 passages.

Intestinal organoid culture
Intestinal crypts were derived from Nudt2fl/fl; R26-CreER (mice and cultured as described before96.

Esophageal cell differentiation
Esophageal epithelial cells were isolated as described above. For experiments including the Nudt21 hypomorph, cNudt21 KO cells were transduced with the inducible HA-Nudt21 virus, selected with blasticidn and pre-treated for 3 days with low dose of of DOX (0.2 µg/ml). For the differentiation assay, cNudt21 KO cells were seeded at 8e3/cm2, and cultured for 4 days in self-renewal media containing either ETOH (Control) or 4OHT (KO) or 4OHT+DOX (Hypomoprh) until a confluency of 8e3/cm2 was reached at day 4. The media was changed every two days. Cells were collected at day 4 for the self-renewal timepoint before initiation of differentiation. Differentiation was induced by first washing the cells with PBS and then changing to CnT-PR-D media (Fisher Scientific) containing 1.8 mM CaCl2. Cells were collected for IF, RNA and protein at days 3 and 6.

Western blot assay
Cells grown in culture were lysed in 200–500 µl of RIPA buffer (Thermo Scientific) supplemented with Complete Mini Protease Inhibitors (Roche), PhosSTOP (Roche), and 0.01 U/µl benzonase (Sigma-Aldrich). The lysates were then incubated on ice for 10 min and sonicated 5 times for 30 s with a 30 s pause in between pulses. The lysates were then cleared for 5 min at maximum speed in a benchtop centrifuge to remove cell debris. The supernatant was boiled together with Laemmli sample buffer (Biorad) and loaded into 5–20% Mini-PROTEAN TGX Precast Protein Gels (Biorad). Protein was transferred to nitrocellulose membranes (Biorad) and blocked for 1 h in 5% powdered milk in TBST (Santa Cruz). Unless otherwise specified, antibodies were used following the manufacturer’s recommendations. Primary antibodies used were: Nudt21 (Santa Cruz, sc-81109, 1:100), β-Actin (Santa Cruz, sc-47778, 1:1000), Cpsf7 (Santa Cruz, sc-393880), Cpsf6 (Bethyl, 50-156-0173), Krt13 (abcam, ab92551), P63 (abcam, ab53039), Seh1l (abcam, ab218531), Nup107 (Invitrogen, PA5-20562), Nup133 (abcam, ab155990), Nup155 (Proteintech, 66359-1-1g), Nup96 (Bethyl, A301-784A), Nup35 (abcam, ab169162), Gapdh (Fisher Scientific, NC0330823), Phospho-ATR (Ser428, Cell Signaling, 2853), ATR (C-1, sc-515173, Santa Cruz). The secondary antibodies used were: goat, anti-rabbit-HRP-conjugated (Thermo Fisher Scientific 31460, 1:2000 dilution) or anti-mouse-HRP-conjugated (Thermo Fisher Scientific 31430, 1:2000 dilution). Proteins were detected using Clarity Western ECL Substrate (Biorad). Images were taken using a ChemiDox Imaging System with enhanced chemiluminescence detection.

Cell cycle and viability assay
Cells were cultured as described above, with specific attention to preventing over-confluence. For the incorporation of EdU in proliferating cells, 1 µM EdU was added to the media and incubated for 1 h in the tissue cell incubator. Epithelial cells were harvested using TrypLE (Gibco), and intestinal organoids were harvested using Trypsin (Thermo Fisher Scientific). Both cell types were fixed and stained according to the manufacturer’s recommendations of the Click-iT Edu-488 kit (Thermo Fisher Scientific). To assess the viability of epithelial cells, they were harvested with TrypLE (Gibco), incubated with 1× Annexin V Binding Buffer (Biolegend, 556454), and stained with FITC-conjugated anti-Annexin V (Biolegend, 640905) and propidium iodide (Thermo Fisher Scientific, BMS500PI) following the manufacturer’s instructions. The gating strategy for EdU-based cell-cycle analysis is shown in Supplementary Fig. 12.

siRNA transfection of epithelial cells
Epithelial cells were cultured as described above. For transfection, cells were seeded at 2e3/cm2 and cultured for one day. On the day of transfection medium was exchanged with fresh pre-warmed medium. The transfection mix was prepared by diluting 30 µl siRNA (20 µM) with 1.1 ml Genmute transfection buffer (SignaGen), followed by brief vortexing and addition of 20 µl Genmute transfection reagent (SignaGen). The mixture was incubated at RT for 15 min and then dropwise added to the cells (4 µl/cm2). For example, 228 µl transfection mixture was added to 10 cm dish containing 10 ml media. Cells were harvested for analysis after three days. All siRNAs were reconstituted in 1× siRNA Buffer (Horizon Discovery) at 20 µM.
ON-TARGETplus Mouse Jmjd1c (108829, Horizon Discovery)
ON-TARGETplus Mouse Sec24a (77371, Horizon Discovery)
ON-TARGETplus Mouse Nup160 (59015, Horizon Discovery)
ON-TARGETplus Mouse Nup107 (103468, Horizon Discovery)
ON-TARGETplus Mouse Seh1l (72124, Horizon Discovery)
siGENOME Non-Targeting siRNA (D-001210-01-05, Horizon Discovery)
siNudt21 Nudt21 (EMU094771, Sigma-Aldrich)

The following siRNAs were purchased as Custom siRNA Standard from Horizon Discovery and pooled at 20 µM for each siRNA.
Nup160_distal_pool:
distal_Nup160_1: AAGCATTTCCTGTAAATCCTA
distal_Nup160_2: AAGAACCGGAGTACGAGTCCT
distal_Nup160_3: AAGGTTCTGTTAGGCTAGGAG
Nup160_Short_pool:
med_isof_Nup160_1: AAGACATCCTTTATCTGCAAA
med_isof_Nup160_2: AACTACCGAAGACATCCTTTA

Immunofluorescence assay (tissue culture cells)
Satellite cells were plated without coating on chamber slides (µ-Slide 8 Well, 80806, ibidi). For epithelial cells, a 30-min 8O4G coating was performed prior to plating. Cells were cultured in their specific media and treated as described in the results section. For immunofluorescence, cells were fixed inside the chamber slides with Paraformaldehyde Solution (PFA), 4% in PBS (AAJ19943K2, Fisher Scientific) for 20 min, followed by three washes with PBS and subsequent permeabilization with 0.5% Triton X-100/PBS (X100-500ML, Sigma-Aldrich) for 5 min. After permeabilization, the slides were washed three times with PBS and blocked for 1 h with 1% BSA/PBS. Primary antibodies were diluted in 1% BSA/PBS, added to the slides, and incubated overnight at 4°C, followed by three washes with PBS. Anti-Nudt21 (sc-81109, Santa Cruz) was diluted 1:100, mAb414 (902901, Biolegend) was diluted 1:1000, Anti-Cytokeratin 13 (ab92551, abcam) was diluted 1:50,000, Trp63/p63 (ab53039, abcam) was diluted 1:1000. Staining with secondary antibodies and DAPI (BD Biosciences, 564907) was performed for 1 h at RT, followed by three washes with PBS. The staining solution contained isoform and species-specific antibodies coupled to Alexa-Fluor-488 (A32766, Thermo Fisher Scientific) and DAPI (both diluted 1:1000 in 1% BSA/PBS). Cells were imaged on a Leica DMi8 Fluorescent microscope (Objective: N PLAN L 40x/0.55, CORR PH2, 11506298).

Nuclear IF signal quantification
Nuclear signal was quantified using Fiji Macro code97. First, a Gaussian filter was applied to the DAPI channel using the function run(“Gaussian Blur…”, “sigma=1”). A mask for nuclei was then created with the following code: setAutoThreshold(“Default dark”); run(“Threshold…”); setThreshold(45, 255); run(“Convert to Mask”). Subsequently, a region of interest (ROI) for each nucleus was generated with the code: run(“Analyze Particles…”, “size=0.1-100 show=Masks display exclude clear include summarize record add in_situ”); run(“Create Selection”). Finally, the desired channel was selected and quantified using roiManager(“multi-measure measure_all”).

RNA FISH assay
Cells were seeded in 8-well chamber slides as outlined in the “Immunofluorescence” section. Treatment conditions are detailed in the “Results” section. For fixation, cells were treated with 4% paraformaldehyde (PFA) for 20 min at RT and subsequently washed three times with phosphate-buffered saline (PBS). For dehydration, cells underwent a graded ethanol (ETOH) series. Cells were incubated in increasing concentrations of ethanol for 1 min each at 30%, 70%, and finally 100%. After the final step, cells were stored in 100% ethanol at -20°C for up to two weeks to ensure preservation. Rehydration was performed by reversing the ethanol concentrations in the same sequence, with each step lasting 1 min, concluding with a PBS wash. Cells were prepared for FISH using the RNAscope™ HiPlex v2 Assay according to the manufacturer’s protocol. The specific probe used was the RNAscope® Probe-PolyA-C3, targeting the poly(A) mRNA tail. For quantification of the poly(A) signal, images were analyzed using a custom Fiji Macro. Initially, the poly(A) channel was duplicated and a Gaussian filter (sigma=2) was applied to enhance feature visibility. The image was then processed to create a binary mask: automated thresholding (Percentile dark), hole filling, watershed algorithm application, and particle analysis (size range 80–1800 square pixels) were performed. For individual cell analysis, selections were created for each cell in an image using the “Create Selection” function. The unaltered poly(A) channel of each ROI was duplicated and saved as a separate TIFF file. Each image file was subsequently opened, and a nuclear mask was applied as detailed in the “Nuclear Signal Quantification” section. Foci quantification within the nucleus and the whole cell was accomplished using the “Find Maxima…” function with a prominence setting of 100. The number of foci detected in the cytoplasm was calculated by subtracting the number of nuclear foci from the total cell detected foci. The notably strong signal within the nucleus suggested active transcription sites, while the cytoplasmic signal predominantly consisted of distinct, clearly visible foci. Cells were imaged on a Leica DMi8 Fluorescent microscope (Objective: N PLAN L 40x/0.55, CORR PH2, 11506298).

3’UTR focused CRISPR screening
Guide RNAs were designed to delete the genomic locus within the 3’UTR that undergoes APA, mimicking the 3’UTR shortening observed in Nudt21 KO. These dgRNA libraries were cloned into a lentiviral plasmid by VectorBuilder (https://en.vectorbuilder.com/vector/Lib240216-1778swe.html). The plasmid harbored two guide RNAs, cloned directly after either the hU6 or macU6 promoter plus scaffold. For selection the plasmid contained and hPGK promotor driving mCherry:T2A:Bsd expression. Lentiviral particles were produced as previously described93. Esophageal basal cells were derived from the iCas9 mouse and transduced with lentiviruses containing dgRNA library at a multiplicity of infection (MOI) of less than 0.1, as assessed by FACS for mCherry. Subsequently, the transduced cells were split into two 15 cm plates: one containing blasticidin (InvivoGen) and dox (treatment), and the other without dox (control). The cells were passaged twice, and genomic DNA was isolated using the Genomic DNA Purification Kit (NEB). The dgRNA sequences were PCR amplified with the primers actatcatatgcttaccgtaacttgaaagtatttcg and agccaaattaattctaattatctctctaacattcttgc, followed by purification using the PCR & DNA Cleanup Kit (NEB). Sequencing was performed by the MGH CCIB DNA Core. Statistical analysis was performed with MAGeCK98.

Satellite cell isolation and in vitro expansion
Satellite cells from control or Nudt21 KO mice treated with tamoxifen (see above), were isolated from mice as previously described37. Satellite cells were seeded on µ-Slide 8 Well, (80806, ibidi) coated with 1 μg/mL collagen type I from rat tail (Sigma) and 10 μg/mL mouse Laminin (Invitrogen) and were cultured for 5 days in F10 (Gibco) supplemented with 20% horse serum (Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin, and GlutaMAX (Thermo), bFGF (5 ng/mL; Sigma) was added daily.

Muscle injury and regeneration
Nudt21fl/fl (control) or Nudt21fl/fl; Pax7-CreER (KO) were treated with tamoxifen as described above. Cardiotoxin (CTX) injury for in vivo regeneration studies was performed by injecting 50 µl of 10 mM CTX (Latoxan) into the tibialis anterior (TA) muscle of animals. Injured TA muscles were collected 14 days post injury and embedded in optimal cutting temperature (OCT) compound (Sakura Tissue-Tek) and frozen in liquid nitrogen-chilled isopentane for 30 s. The frozen muscles were then sectioned using a cryostat set at -20°C to obtain 10 μm thick mid-belly cross-sections. For dystrophin and Hoechst double staining, the mid-belly sections were first fixed with 1% paraformaldehyde (PFA) in PBS for 10 min at room temperature (RT). Following fixation, the sections were washed with PBS three times. The sections were blocked using a staining buffer composed of 0.5% BSA and Triton-X100 in PBS for 20 min at RT. After blocking, the sections were incubated with primary antibodies in the staining buffer for 1 hour at RT. The primary antibody used was anti-dystrophin (Mandys8, Santa Cruz, sc-58754) at a dilution of 1:100. Following primary antibody incubation, the sections were washed with three times with PBS. Subsequently, the sections were incubated with secondary antibodies in the staining buffer for 1 h at RT. The secondary antibody used was anti-mouse IgG2b Alexa 594 (Thermo Fisher Scientific) at a dilution of 1:250. After secondary antibody incubation, the sections were washed with PBS three times. The sections were then stained with Hoechst dye at a dilution of 1:1000 in PBS. Finally, the sections were washed with PBS, repeated two times, and prepared for imaging.

Complete blood counts with differential
Whole blood was collected via retro-orbital bleeding using heparinized micro-hemocrit capillary tubes (Thermo Fisher Scientific) and transferred into EDTA-coated microvette tubes (Sarstedt). Complete blood counts with differential were then analyzed using an Element HT5 machine (Heska).

Flow cytometry (bone marrow)
Whole blood was processed for flow cytometry by lysing red blood cells in RBC lysis buffer (BioLegend) for 8 min on ice. Cells were then centrifuged at 1200 rpm for 5 min, resuspended in FACS buffer (2% fetal bovine serum (Hyclone) in PBS), and filtered through a 0.2 μm mesh. After another centrifugation at 1200 rpm for 5 min, the cells were stained on ice for 1 h. The following antibodies were used: CD3e (APC; BD Biosciences; clone 145-2C11), CD45.1 (PE; BioLegend; clone A20), CD45.2 (Pacific Blue; BioLegend; clone 104), Mac1 (FITC; eBiosciences; clone M1/70), B220/CD45R (APC eFluor 780; Life Technologies; clone RA3-6B2), Gr1 (PE or PE/Cy5; eBiosciences; clone RB6-8C5), Fc block (BD Biosciences), propidium iodide (BD Biosciences), and DAPI (BD Biosciences). After staining, cells were washed with FACS buffer. Propidium iodide or DAPI was used for viability staining. Bone marrow was harvested from euthanized mice by flushing femurs and tibiae with FACS buffer using a 27½ gauge needle, followed by dissociation with a 22 gauge needle. Cells were centrifuged at 1200 rpm for 5 min, resuspended in 5 ml of RBC lysis buffer for 8 min on ice, and the lysis was quenched with 20 ml of culture media before another centrifugation at 1200 rpm for 5 min. Cells were resuspended in 10 ml of FACS buffer, filtered through a 0.2 μm mesh, and counted. Cells were aliquoted and stained for the detection of HSPCs with the following panel: SLAM panel (3 million cells): Lineage markers [B220/CD45R (PE/Cy5; BioLegend; clone RA3-6B2), Ter119 (PE/Cy5; eBiosciences; clone TER-119), CD3e (PE/Cy5; BioLegend; clone 145-2C11), Mac1 (PE/Cy5; eBiosciences; clone M1/70), Gr1 (PE/Cy5; eBiosciences; clone RB6-8C5)], Sca1 (PE/Cy7; eBiosciences; clone D7), CD150 (PE or BV605; BioLegend; clone TC15-12F12.2), CD48 (APC; BioLegend; clone HM48-1), CD45.1 (PE; BioLegend; clone A20), CD45.2 (Pacific Blue; BioLegend; clone 104), cKit (eFluor 780; eBiosciences; clone 2B8), Fc block (BD Biosciences). DAPI was used as a viability dye.

Hematopoietic progenitor in vitro culture
Ten thousand LSK cells were sorted and cultured in IMDM (Gibco) supplemented with 10% FBS, 1× GlutaMax (Life Technologies), 100 U/mL Penicillin, 100 μg/mL Streptomycin (Life Technologies), and 50 μM β-mercaptoethanol (Life Technologies). 10 ng/ml IL3 (Peprotech), 10 ng/ml IL6 (Peprotech), 10 ng/ml SCF (Peprotech), 5 ng/ml GM-CSF (Peprotech), 50 ng/ml TPO (Peprotech), and 3 U/ml EPO (Bio legend) were added. Where indicated, 4OHT (0.05 µM) or ETOH was added.

PAS-seq analysis
Total RNA from cells was isolated with the RNeasy Kit (QIazol). PAS-seq library preparation and data analyses were performed as previously described99. APA events were considered significant if they met both criteria: a false discovery rate (FDR) < 0.05 and a change in distal PAS usage greater than 14.5%. Statistical significance was assessed using edgeR, and FDR values were calculated using the Benjamini–Hochberg (BH) method to correct for multiple testing.

Single-cell RNA-seq data analysis
Chromium single cell 3′ reagent v3.0 kit (10x Genomics), followed by sequencing on Illumina NextSeq 2000 instrument, which resulted in approximately ranging from 250 to 400 million reads per sample. Primary processing of scRNA-Seq sequencing Single-cell RNA-seq library construction was performed on the Chromium 10× instrument using data was performed using Cell Ranger (v6.0.0) (https://support.10xgenomics.com/single-cell-gene-expression/software/overview/welcome) [https://doi.org/10.1038/ncomms14049]. In brief, reads were aligned to mm10 mouse reference genome (mapping rate of ~95%), followed by the generation of read counts per gene in each cell. Further analysis was performed using the Seurat 3.2.3 package (https://satijalab.org/seurat/). We filtered out cells with <200 expressed genes and genes expressed in <3 cells, followed by the exclusion of cells with high content of mitochondrial transcripts (>10% of total reads). Counts across all cells for each sample were normalized using the NormalizeData function in Seurat. Using the FindVariableFeatures function, 2000 features were selected to be used in principal component analysis (PCA). All individual samples were integrated using Seurat canonical correlation analysis (CCA) method. Integration anchors were determined using FindIntegrationAnchors function and then used in IntegrateData function. UMAP plots and cell clusters were generated using RunUMAP and FindClusters functions, followed by manual cell type annotation based on type-specific gene markers.

RNA-sequencing and bioinformatic analysis
mRNA-seq libraries were constructed with the SMARTer v3 kit (Clontech) and sequenced on the Illumina HiSeq 2500 as PE50, producing approximately 20–30 million reads per sample. microRNA-seq libraries were constructed with the NEBNext® Small RNA Library Prep Set (NEB) and sequenced on the Illumina HiSeq 2500, producing approximately 10 million reads per sample as SR50. Transcriptome mapping was performed using STAR with the UCSC annotation of the mm10 reference genome. To align microRNAs to the reference database miRbase (mature.fa). The mature.fa file was U to T converted via: “sed ‘/^[^>]/s/U/T/g’ mature.fa > mature_conv.fa”. And aligned with bowtie100 using the following attributes “-v1 -a --best –strata”. Read counts for individual genes were generated using featureCounts from the Subread package101. microRNA counts were generated with samtools idxstats. Differential expression analysis for mRNA and microRNA was carried out with EdgeR after normalizing read counts and including only genes with CPM > 1 in at least one sample. Differentially expressed genes or microRNAs were identified based on a >2-fold change in expression value, unless otherwise specified.

Proteomics
Protein concentration of cell lysates was determined by DC Assay (Bio-Rad). Reduction alkylation, precipitation and TMT labeling and Liquid chromatography coupled to mass spectrometry were performed as previously described51. Differential analysis was performed by the DEP package102.

Translational efficiency
Conditional Ribosome profiling cDNA library preparation for deep sequencing was performed as previously described90. Translation efficiency (TE) was calculated using ribosome protected fragments (RPF) and RNA-seq reads per kilobase per million mapped reads (RPKM). TE = (RPKM[RPF]/RPKM[mRNA]). We obtained transcript, CDS and 3′UTR length for mouse genes from UCSC (GRCm38_UCSC_refGene.gtf).

IVT mRNA synthesis and transfection
The mRNA constructs were generated by VectorBuilder using a standardized workflow. Briefly, Nup160 CDS fused N-terminally to eGFP (or eGFP alone) and carrying either Short, Mid, or Long Nup160 3′UTRs were cloned into pmRVac plasmid backbones under control of a T7 promoter. Following sequence validation, plasmids were linearized and used as templates for in vitro transcription with complete substitution of uridine by N1-methylpseudouridine (m1Ψ), co-transcriptional Cap 1, and poly(A) addition. The resulting mRNAs were purified, quality-controlled, and delivered as RNase-free, ready-to-use solutions.
mRNA transfection was performed with MessengerMAX Transfection Reagent and Opti-MEM Reduced Serum Medium (both purchased from Thermo Fisher Scientific). One day before transfection (day -1), cells were seeded at 3×104 cells per well in 24-well plastic or glass-bottom plates. Equimolar concentrations of distinct IVT mRNAs were used for every transfection, with maximum mRNA dose not exceeding 25 ng per 1×104 cells. On the day of transfection (day 0), culture medium was exchanged with pre-warmed culture media 30 min prior transfection. The transfection mixture was prepared in two separate tubes. In the first tube, IVT mRNAs were diluted in 50 µl OPTI-MEM and left at room temperature. In the second tube, the transfection reagent was prepared by diluting 1.5 µl MessengerMAX in 50 µl Opti-MEM followed by brief vortexing (2–3 s). Both reactions were incubated separately at room temperature for 10 min before they were combined. Both reactions were incubated for 5 min at room temperature and combined followed by an additional 10 min incubation at room temperature. Before transfection, the brought 900 µl Opti-MEM were added to the mix. The resulting mRNA–lipid complexes were added to cells dropwise to ensure even distribution. For 24-well plates, 150–300 µl of complex were dispensed per well. Transfected cells were maintained under standard culture conditions. On day 1, cells were gently rinsed with PBS to remove phenol red and examined microscopically to assess cell health and transfection performance prior to downstream procedures. eGFP Expression was assessed by microscopy or FACS 24h post transfection.

RNA-IP/MS
N-Terminally Biotinylated RNAs were purchased from VectorBuilder and all reagents for the RNA-immune precipitation were from Thermo Fisher. For RNA-IP, Biotinylated IVT RNAs were separately resuspended in equimolar concentrations of 1 µM in 500 µl ice-cold binding buffer (20 mM Tris-HCl pH 8.0, 250 mM NaCl, 1 mM EDTA, RNase inhibitor) and denatured at 65°C for 10 min, followed by cooling on ice. For coupling, 25 µl Dynabeads MyOne Streptavidin C1 (Thermo Fisher) were washed 5× in 1 ml washing buffer (20 mM Tris-HCL pH 8, 250 mM NaCl, 50 mM KCl, 2.5 mM MgCl, RNase inhibitor, protease inhibitor) and incubated for 1–2 h at 4°C with rotation. After washing, RNA-bead complexes were incubated with cell lysates. Cell lysates were prepared by resuspending 10⁷ cells in 1 ml lysis buffer (20 mM Tris-HCl pH 8.0, 250 mM NaCl, 50 mM KCl, 2.5 mM MgCl2, 0.5% Triton X-100, 0.2% NP-40, RNase inhibitor, protease inhibitor, 0.6% RNase-free DNase), followed by 3x needle passaging and sonication (Bioruptor Pico; 30 s on/off, 5 cycles at 4°C) and clarification through centrifugation. Cell lysate supernatants were combines with RNA-bead complexes and incubated for 4 h at 4°C with rotation. Beads were washed in 3× wash buffer and 1× rinse buffer (20 mM Tris-HCL pH 8, 250 mM NaCl, 50 mM KCl, 2.5 mM MgCl2) to remove residual inhibitors. Bound proteins were eluted in 200 µl elution buffer (10 mM Tris-HCl pH 8.0, 15 mM NaCl containing 0.1 mg/mL RNase A) for 10 min at room temperature followed by 20 min at 30°C. Beads were removed via and eluates were transferred to fresh tubes, further incubated on ice, and stored at –80°C until submitted for label-free mass spectrometry.

ceRNA analysis
This analysis was carried out as reported previously42. Briefly, the datasets PAS-seq (APA), bulk RNA-seq (three biological replicates per condition: Ctrl, Nudt21 KO, Hypomorph) and small-RNA-seq (miRNA) were used to determine putative ceRNAs. For each miRNA, we quantified all unordered RNA-RNA pairs among its targets. To ensure robustness, only genes with >20 distinct targeting miRNAs were further considered. Pairwise Pearson correlations were computed across the three replicates. Pairs with r>0.6 were classified as ceRNAs.

Integrative analysis
The list of essential genes was derived by combining two datasets: CRISPR_Inferred_essential (23q4) and CRISPR_common_essentials (22q2), both available from the DepMap portal (https://depmap.org/portal/data_page/). miRNA targets were identified by extracting annotated miRNA-mRNA interactions from TarBase103, specifically using the Mus_musculus_TarBase_v9.tsv file. Only miRNA-mRNA interactions positioned at the 3’UTR with a stringent microT score ≥0.7, as previously recommended104, were considered. We collected protein complex information from the EBI databases to compile a comprehensive dataset of known protein complexes. All processed ‘Omics’ data are available as tables in the Supplementary Data.

Disome selective profiling (DiSP)
Cells were lysed and subjected to RNase1 digestion, ribosome-nascent chain complexes were separated to disome (D) and monosome on sucrose density gradient91,105. Ribosome profiling reads from D and monosome (M) fractions were processed using a custom R pipeline. For each coding sequence (CDS), aligned reads were mapped to the nucleotide grid and log2(D/M) ratios were computed. To quantify positional changes in co-translational assembly (co-co-assembly), each CDS was divided into three windows: early (5–25%), mid (30–70%), and tail (last 30%), with enforced minimum lengths. For each window, reads were summed, and window-level log₂(D/M) were calculated. The co-co assembly score was defined as the maximum of (mid–early) or (tail–early), capturing either mid-body or C-terminal enrichment.
Genes were excluded if they lacked sufficient CDS coverage, had fewer than the required number of codons per window, or failed adaptive read-support thresholds (scaled to library depth). This pipeline was applied to Nudt21 knockout (KO) and control (WT) datasets (two replicates each). Of ~13,800 coding sequences, ~6400–7300 genes per replicate passed filtering (52–56% retention), consistent with lower sequencing depth compared to benchmark HEK293T data. Replicates showed moderate reproducibility (Spearman ρ≈0.43; ~50% overlap among the top 200 genes). Consistent with Bertolini et al.91, genes encoding transmembrane domain (TMD) proteins were excluded, as membrane proteins can yield false-positive DiSP signals due to aberrant sedimentation. For KO vs WT comparison, mean co-co assembly scores were computed per gene, and the difference (Δ= KO−WT) was used as the effect size.

Statistical analysis
Statistical evaluation was performed with GraphPad Prism 5 or R using linear regression and two-tailed tests: t-test, Anova test or Wilcoxon rank test as specified in the Figure legends. The number of independent data points n always refers to biological replicates. Bonferroni correction was applied to the gene expression analysis to correct for multiple comparisons. *, p≤0.05; **, p≤0.01; ***, p≤0.001, n.s., not significant, p>0.05.
Statistical analyses were conducted using Prism software (GraphPad) and R software. Specific details for each analysis, including the number of replicates, are provided in the Figure legends. Analyses were performed comparing genotypes and treatment conditions (wild-type, Nudt21 knockout, and hypomorph). Where applicable, experimental replicates and time points were included as covariates in linear models. Sex was balanced across groups (50% male, 50% female), but it was not included as a covariate, as no sex-specific effects were anticipated or observed. No additional covariates were tested.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Tsopoulidis et al. examined the functional impacts of Nudt21 on cell proliferation/self-renewal and\ndifferentiation using mouse models. The Hochedlinger group, by using shRNAs and siRNAs, previously reported a\nsignificant role of Nudt21 in cell differentiation and dedifferentiation in the context of induced pluripotent cells. Here they use\nin vivo systemic or cell type-specific (basal stem cells of squamous epithelia) knockout (KO) mice as well as KO cells with\nvaring levels of Nudt21 expression through inducible expression of exogenous Nudt21. The major finding is that Nudt21\nexpression at different levels can exert distinct functions on cell proliferation/self-renewal and differentiation. In KO cells, cell\nproliferation is inhibited. At the molecular level, transcripts with miRNA target sites are upregulated, in line with the notion\nthat 3’UTR shortening caused by Nudt21 deficiency mitigates miRNA targeting. In hypomorphs with ~70% reduction of\nexpression, the authors found less potent 3’UTR shortening and weaker the miRNA effect. Interestingly, while the\nhypomorphs have limited effects on cell proliferation/self-renewal, cell differentiation is affected, indicating that\nproliferation/self-renewal can be uncoupled from differentiation. Moreover, the authors found that many protein complexes\nare downregulated in KO cells, such as the nuclear pore complex whose expression and functions were specifically\nexamined in this work. The authors suggest that 3’UTRs may play roles in co-translational complex assembly. Overall, this\nstudy was carried out with good rigor. The conclusions are somewhat expected based what have already been known. But\nthis work does add useful information, especially with the in vivo data, on how Nudt21 exerts its functions. I have only some\nminor comments and suggestions.\n- The authors speculate that the suppression of protein complex expression and formation in Nudt21 deficient conditions\nmight be due to defects in 3’UTR-mediated co-translational assembly. This is an interesting hypothesis. However, it might\nalso be possible that some chaperone proteins responsible for protein complex assembly are particularly sensitive to 3’UTR\nsize regulation. The authors may want to discuss this as well.\n- Some previous studies indicated that global 3’UTR shortening could lead to alteration of miRNA targeting through\ncompeting endogenous RNA (ceRNA) networks. It would be informative and helpful to the field if the authors could use their\ndata to evaluate this aspect of APA function.\n- Fig. 3f, y-axis, it would be more appropriate to use ‘shortened’ instead of ‘short’ 3’UTRs. Likewise, ‘lengthened’ should be\nused in place of ‘long’.", "reviewer_2": "In this manuscript, the authors explore the role of NUDT21 in stem cell renewal and differentiation in a mouse model system.\nInitially, the uncover a gradient of NUDT21 expression in various stem cell / differentiated cell populations using IF.\nLeveraging this observation, they create a NUDT21 conditional knockout mouse. Inducing knockout either ubiquitously or in\nbasal stem cells and squamous epithelia caused a quick morbidity in adult mice (<7days), which is a novel finding in of\nitself. They go onto show that depletion of NUDT21 causes a cell cycle defect in these cell types and that satellite cells that\nhave exited the cell cycle do not show such a phenotype. The results indicating that despite the lack of phenotype in satellite\ncells can be upended upon acute muscular injury is highly compelling. They then develop a dox-inducible NUDT21\nexpression system to carefully titrate expression allowing for a ‘hypomorphic’ state to be achived in order to show\nuncoupling of renewal from differentiation. Within this system, they conduct PAS-seq to identify key pre-mRNA targets of\nNUDT21 that undergo shifting upon depletion and they validate those with siRNA-depletion in order to rescue. They then\nreport that reduction in NUDT21blocks differentiation through the loss of miRNA repression of specific target genes yet the\nblock to self-renewal is due to a more complicated effect: loss of cotranslational accumulation of multiprotein complex\nencoding mRNA product proteins.\nOverall, this is an outstanding study and provides novel insight at multiple levels. I only have several minor concerns\nto be addressed to support publication.\n1. In terms of the hematopoietic stem cell/differentiated cell expression shown for NUDT21 in Figure 1c; can the authors also\ninvestigate NUDT21 expression during human hematopoietic differentiation by tapping into the Bloodspot database? This\nwould add depth to the observations by providing further confirmation of human expression.\n2. Can the authors describe how old mice were when they received tamoxifen in Figure 1f? It is a compelling and novel\nresult that adult mice die quickly upon deletion, but the definition of ‘adult’ was not made clear. In addition to making this\nmore clear, can the authors rationalize/justify why they selected this particular age to cause acute loss of NUDT21?\n3. In figure 3b, the authors reference that hypomorphic cultures can grow 8 cell divisions in the text but it is not clear where\nthat data is in the actual figure. Appears to describe percentage of cells in S phase. Can the authors clarify?\n4. For the PAS-seq in Figure 3c, can the authors clarify the statistical values used to call 3’UTR shortening versus\nlengthening? While it might be in the legend, definining this in the results is important for 3’-end specialists who would\nread this paper.\n5. I leave it to the authors discretion, but an experiment to further support conclusions derived from Figure 3j, transfecting\nantagomirs to miR-449a-5p and miR-30d-5p predicting leading to a phenocopy result would be strong to support their role.\n6. Can the authors clarify the rationale for filtering proteins through the essentialome? Specifically, they refer to filtering\nproteins from 353 to 73 for final analysis – but shouldn’t they be stating that filtering resulted in a list of 73 essential proteins\nor 73 non-essential proteins (I believe it is the former, not the latter)? In other words, the use of the word ‘filter’ is somewhat\nvague and makes it not immediately clear as to what filter resulted in. The figure makes it more clear, but the text can be\nimproved.\n7. Along these same lines, please consider changing the sentence: “Importantly, protein levels for the 73 candidate\nESSENTIAL genes decreased at day 4 even though…”\n8. The result that these 73 genes are enriched in multi-protein complexes is also quite compelling – but it is not clear if this\nobservation is conserved in human. This seems easy enough to determine as the authors can just compare these\ngenes/gene structures to their human counterparts and query PADB. This would further strengthen their general thesis.\n9. The results in Figures 4/5 demonstrate the 3’UTR shortening of specific nucleoporin mRNA result in their loss of protein\nexpression despite no change in mRNA stability or translational competency. The authors posit that 3’UTR-mediated co-\ntranslational assembly as a possible explanation for this result. While I believe testing this mechanistic hypothesis is beyond\nthe scope of this paper, it is not unreasonable to suggest to the authors that heterologous reporter experiments using\nluciferase-3’UTRs would be beneficial. In other words, if the protein encoded by the short, medium, or long nucleoporin\n3’UTR is no longer a nucleoporin and is not in need of cotranslational assembly, will its expression/accumulation no longer\nbe attenuated??", "reviewer_3": "Tsopoulidis et al., generated conditional KO mice for Nudt21, the most important regulator of APA. This KO, together with a\nNudt21 ‘hypomorph’, which was obtained by rescuing the KO with approximately 25% of Nudt21 expression allows the\nauthors to investigate the biological consequences of impaired APA. The authors observed that full deletion of Nudt21 in\nadult mice results in their lethality within 5-7 days. This is a striking finding and demonstrates the biological importance of\nAPA. As Nudt21 is highly expressed in the stem cell compartment, the authors then focused on stem cell biology to\ninvestigate how the loss of Nudt21 results in animal death. They observed that highly proliferative cell types, such as the\ngastrointestinal tract or hematopoietic system are strongly affected, whereas tissues without regeneration are not affected,\nbut become affected during regeneration stress. All these aspects were very nicely demonstrated, which is the strength of\nthe paper. These findings should definitely get published and are very interesting for the community.\nThe authors then try to determine the molecular mechanism by which loss of Nudt21 affects proliferation (cell cycle) or\ndifferentiation. They perform PAS-seq and mass spectrometry analysis and pursue different hypotheses, including\nmiRNA regulation and loss of nuclear pores as the potential underlying mechanisms. However, these investigations are not\nperformed very rigorously and are only modestly convincing.\nSpecific points:\n1. The miRNA analysis does not make the paper stronger. It is only correlative and does not add real insights. Even if\nloss of miRNA regulation would cause slight upregulation of some of the factors shown in Fig. 3i, it is unclear whether\nthis has any relevance for the phenotype observed. In my opinion, the paper would benefit from leaving this part (Fig. 3f-3l and Ext Data\nFig. 4f-4g) out as it does not add anything to the proposed mechanism by which loss of Nudt21 causes death.\n2. Nudt21 affects the nuclear pore.\nIt is currently unclear, what the authors think is the molecular mechanism by which Nudt21 loss affects the nuclear pore. If\nNup160, Nup107 and Seh1L are essential genes and they are knocked-down, it is not surprising that the cells show\ndecreases in cell cycle progression. Moreover, the figure with the chromatin bridges in Ext Data Fig. 5j, k should be\nremoved. If the authors think that formation of the chromatin bridges are causative, it needs to be demonstrated.\nIf they think that decreased Nup160 protein levels are causative for the phenotype, it is important to restore Nup160\nprotein level to investigate if this rescues some of the phenotypes seen in Nudt21 loss of function cells.\nOr, if the authors think that the longest 3’UTR isoform of Nup160 is responsible for protein complex assembly with other\nproteins in the nuclear pore, then, direct evidence needs to be shown. At the current state of the manuscript, lots of data are\npresented, but the overall message of this part is not clear.\n3. The data in Fig. 4g showed upregulation of 26S proteasome complexes in the KO. As an alternative explanation, could\nthis contribute to the observed protein destabilization, and therefore not all observed decreases in protein levels are direct\nconsequences of APA-dependent protein complex formation? Importantly, most essential genes, not just genes regulated by\nNudt21 through APA, are downregulated in the KO, which further raise the question of the direct causal relationship between\nits role in APA and the phenotype.\nIn the critical Fig./Extended Fig 5, which should help to establish causal relationship between APA and the phenotype, there\nare some problems: (1) Regarding phenotype in mRNA export, images in Nup160 long isoform deletion is inconsistent with\nwhat is shown in the quantification. I think a better way of presenting the result is to plot the fraction of mRNA exported/cell,\nrather than the absolute number. (2) The siRNA targeting the middle isoform is not isoform-specific and leads to equal\ndecrease in the mRNA level of the longest isoform and of Nup160 protein levels.\n4. A strength of the paper is the genome-wide and proteome-wide analyses in a time course of Nudt21 depletion. As the\nauthors seem to have problems of pinpointing to the mechanism responsible for the observed phenotype, it is essential to\nprovide the entire dataset for other researchers to use (as supplementary table). This should include all the data on APA, all\nthe mass spectrometry data (not just the data on the essential differentially expressed proteins). Also, the data on the\nhypomorph should be included in this table. If this table is presented well (with p-values and fold-changes), it would be a\nuseful resource for the community to test alternative hypotheses on the mechanism by which Nudt21 has such strong effects\non self-renewal and differentiation.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "We thank this reviewer for their overall interest and conclusion that our study was performed with good rigor\nand adds useful information to the field.\n- The authors speculate that the suppression of protein complex expression and formation in Nudt21 deficient\nconditions might be due to defects in 3’UTR-mediated co-translational assembly. This is an interesting hypothesis.\nHowever, it might also be possible that some chaperone proteins responsible for protein complex assembly are\nparticularly sensitive to 3’UTR size regulation. The authors may want to discuss this as well.\nWe thank the reviewer for suggesting this alternative interpretation. To test whether chaperones are directly\nimpacted by Nudt21 loss, we examined 3′UTR shortening and protein abundance of known chaperones in\nNudt21 KO cells. Among the 12 chaperones detected in our proteomic data, only three (Dnaja1, Hsp90aa1,\nand Hspa1a) showed 3′UTR shortening upon Nudt21 KO. Importantly, none of these chaperones was\nsignificantly downregulated at the protein level, arguing against a direct effect of 3’UTR shortening on\nchaperone destabilization in Nudt21 KO cells.\nHowever, we note that certain chaperones were upregulated in Nudt21 KO cells even though their\ncorresponding mRNAs did not exhibit 3′UTR changes, including components of the “chaperonin-containing\nT-complex” and “protein folding chaperone complex”. These upregulated proteins likely reflect secondary\nchanges caused by failed protein complex assembly rather than direct 3′UTR-dependent regulation of\nchaperones themselves. These results have been added to the text (page 10, lines 424-440) and to\nExtended Data Figure 7 copied below.\nExtended Data Fig.7: Proteotoxic stress upon Nudt21 KO\na: GO analysis for proteins upregulated in Nudt21 KO cells\nb: Differential protein expression of chaperones with unsupervised clustering\nc: GO analysis for clusters in (b)\nd: Genes ranked by their PAS change (proximal to distal PAS usage) ratio upon Nudt21 KO. Highlighted are chaperones shown in\n(b)\ne: Effect of ISRIB (100µM) on cell expansion. 1E5 cells were plated on a 10cm dish and cell number was counted after 6 days of\ntreatment.\n- Some previous studies indicated that global 3’UTR shortening could lead to alteration of miRNA targeting through\ncompeting endogenous RNA (ceRNA) networks. It would be informative and helpful to the field if the authors could\nuse their data to evaluate this aspect of APA function.\nWe thank the reviewer for suggesting to assess ceRNAs in our model. To this end, we determined the global\nceRNA network by grouping mRNAs with similar miRNA-binding sites. RNAs within these groups were\ndefined as ceRNAs if they were transcriptionally co-regulated (>0.6 Pearson’s correlation coefficient)1 . In\ncontrol cells, we found that 41.9% of mRNA pairs behaved as ceRNAs based on these criteria. In Nudt21\nKO cells, however, the abundance of ceRNAs decreased to 27.7%. In hypomorphic cells, 36.3% of mRNA\npairs behaved as ceRNAs consistent with a dose-dependent effect. Our observations support previous\nmodels according to which the loss of 3’UTRs results in disrupted ceRNA networks1 .\nDeregulation of ceRNA networks in Nudt21-depleted cells is exemplified by the APA target Sec24a. Sec24a\nis critical for the Nudt21-dependent differentiation defect, and its long APA isoform contains 28 predicted\nmiRNA binding sites (see updated Figure 3i). In control cells, where Sec24a predominantly produces long\n3’UTR isoforms, we find that the expression of predicted ceRNAs is significantly correlated with the\nexpression of Sec24a, suggesting strong co-regulation via shared miRNA pools. However, upon Nudt21\ndepletion in either KO or hypomorphic cells, this correlation is disrupted. This observation is thus consistent\nwith the interpretation that loss of the long 3’UTR isoform of Sec24a disrupts a crucial ceRNA network by\naltering the corresponding miRNA target landscape. More generally, these results support the notion that\nAPA modulates miRNA-mediated post-transcriptional gene regulation. These results have been added to the\ntext (lines 353-373 of the text, pages 8-9) and to Extended Data Figure 5, copied below.\nExtended Data Fig. 5: Nudt21 modulation perturbs global ceRNA co-regulation\na-c: Distribution of pairwise Pearson correlations for mRNA pairs that share experimentally supported miRNA binding. Pairs were\nbuilt from all genes targeted by the same miRNA. For robustness, only RNAs with > 20 distinct targeting miRNAs were further\nconsidered ceRNAs. Correlations were computed across biological replicates (n = 3 per condition). Red line marks the ceRNA\nthreshold (r = 0.6). The proportion of pairs above this threshold is considered to be ceRNAs and indicated as a percentage (%) of all\npairs.\nd-f: Example ceRNA network centered on Sec24a. Each small panel shows replicate-level scatter of Sec24a vs. a miRNA-co-\ntargeted mRNA\n- Fig. 3f, y-axis, it would be more appropriate to use ‘shortened’ instead of ‘short’ 3’UTRs. Likewise, ‘lengthened’\nshould be used in place of ‘long’.\nWe fully agree with this suggestion and have accordingly revised the y-axis label in Fig. 3f to use ‘shortened’\nand ‘lengthened’ 3’UTRs.\nFigure 3: Modulation of CFIm levels uncouples self-renewal from differentiation\nf: Euler plot showing common and distinct changes to PAS usage when comparing Nudt21 KO vs control and Nudt21 hypomorph vs\ncontrol. Commonly shortened 3’UTRs (944 mRNAs) were used for subsequent analysis (g-j).\nWe thank this reviewer for noting that our study is outstanding and novel at several levels.\n1. In terms of the hematopoietic stem cell/differentiated cell expression shown for NUDT21 in Figure 1c; can the\nauthors also investigate NUDT21 expression during human hematopoietic differentiation by tapping into the\nBloodspot database? This would add depth to the observations by providing further confirmation of human\nexpression.\nWe appreciate the reviewer’s suggestion and have now queried the Bloodspot database to evaluate\nNUDT21 expression during human hematopoietic differentiation. Consistent with our mouse data,\nNUDT21 expression is highest in human hematopoietic stem and progenitor cells (HSPCs) and\ndecreases upon differentiation into mature cell types. These data support the conclusion that NUDT21’s\nrole in early hematopoiesis is conserved across species. A summary of the Bloodspot expression profile\nhas been added to Extended Data Fig.2a and is referenced in the main text on pages 3-4, lines 134-137\nof the text.\nExtended Data Fig.2: CFIm is essential for the regeneration of epithelial tissues and the hematopoietic system\na: NUDT21 expression in the human hematopoietic system, based on the Bloodspot database2 .\n2. Can the authors describe how old mice were when they received tamoxifen in Figure 1f? It is a compelling and\nnovel result that adult mice die quickly upon deletion, but the definition of ‘adult’ was not made clear. In addition to\nmaking this more clear, can the authors rationalize/justify why they selected this particular age to cause acute loss of\nNUDT21?\nWe apologize for not clearly defining “adult” mice in the manuscript. In Figure 1f, we used mice between 3\nand 6 months of age at the time of tamoxifen administration. This information has now been added to\nMaterials and Methods (page 51, lines 1183-1184 of the text), Figure 1 legend (line 729 of the text, page 17),\nand the main text. We selected this age because mice at 3–6 months of age are considered adult and\nexhibit stable tissue homeostasis without confounding effects of aging. This allowed us to assess the\nconsequences of acute Nudt21 loss in a physiologically homeostatic adult context, rather than in developing\nor aged animals.\n3. In figure 3b, the authors reference that hypomorphic cultures can grow 8 cell divisions in the text but it is not clear\nwhere that data is in the actual figure. Appears to describe percentage of cells in S phase. Can the authors clarify?\nWe apologize for this vague statement, which we have now clarified. Briefly, we observed that hypomorphic\ncultures can be passaged for at least 8 passages and in our experience, each passage entails at least one\nround of population doubling/cell division. However, we have never precisely measured these parameters\nand therefore modified the relevant sentence as follows: \"Leveraging this system, we determined that a\nreduction of Nudt21 levels down to ~25% of control levels using 50ng/ml dox is sufficient to support stem cell\nself-renewal and cell cycle progression (Figure 3b top), for at least 8 passages (data not shown).\" (lines 258-\n261 of the text, page 6).\n4. For the PAS-seq in Figure 3c, can the authors clarify the statistical values used to call 3’UTR shortening versus\nlengthening? While it might be in the legend, defining this in the results is important for 3’-end specialists who would\nread this paper.\nWe thank the reviewer for raising this important point, which we have addressed in the revised version of the\nmanuscript. Briefly, for our analysis of PAS-seq data (Figure 3d and e), we considered an APA event to be\nsignificant if it had a false discovery rate (FDR) < 0.05 and showed a change in distal PAS usage > 14.5%.\nStatistical significance was assessed using edgeR, and FDR was calculated using the Benjamini-Hochberg\n(BH) method to correct for multiple testing. We have added this information to the figure legend of Figure 3\n(lines 785-789 of the text, page 21) and materials and methods (lines 1583-1589 of the text, page 61) as\nsuggested.\n5. I leave it to the authors discretion, but an experiment to further support conclusions derived from Figure 3j,\ntransfecting antagomirs to miR-449a-5p and miR-30d-5p predicting leading to a phenocopy result would be strong to\nsupport their role.\nWe thank the reviewer for suggesting this experiment to further strengthen the role of miRNAs in the\ndifferentiation block. To this end, we transfected antagomirs targeting miRNAs predicted to bind the 3′UTRs\nof Sec24a and Jmjd1c, including miR-449a-5p, miR-30d-5p, and miR-34a-5p into esophageal stem cells. Of\nthese, only anti-miR-34a, which targets Jmjd1c, was able to delay differentiation akin to Nudt21\nhypomorphs, as indicated by elevated p63 expression. The other antagomirs were either unable to\nphenocopy hypomorph cells or decreased p63 levels, indicating that interfering with single miRNA–mRNA\ninteractions may be insufficient to fully recapitulate the effects of Nudt21 hypomorph cells. These results are\nconsistent with the notion that Sec24a and Jmjd1c are hubs of miRNA regulation / ceRNA networks and\ninterference with one of their cognate miRNAs is not sufficient to increase transcript stability. These results\nhave been added to Extended Data Fig. 4 and are discussed on page 8, lines 342-351 of the text.\nExtended Data Fig.4: Modulation of Nudt21/CFIm levels rewires PAS usage and uncouples proliferation from differentiation\nh: Anti-miR-induced effect on Trp63 expression in esophageal stem cells. Wild-type esophageal stem cells were transfected with\ncontrol anti-miR (antiMirCtrl) or anti-miRs targeting miRNAs predicted to regulate Jmjd1c (miR-27, miR-34a, miR-449) or Sec24a\n(miR-15b, miR-23a, miR-30a, miR-30d). Nuclear Trp63 protein levels were microscopically quantified.\n6. Can the authors clarify the rationale for filtering proteins through the essentialome? Specifically, they refer to\nfiltering proteins from 353 to 73 for final analysis – but shouldn’t they be stating that filtering resulted in a list of 73\nessential proteins or 73 non-essential proteins (I believe it is the former, not the latter)? In other words, the use of the\nword ‘filter’ is somewhat vague and makes it not immediately clear as to what filter resulted in. The figure makes it\nmore clear, but the text can be improved.\nWe thank the reviewer for pointing out this confusing statement, which we have clarified in the revision. The\nreviewer is absolutely correct in that our filtering resulted in a list of 73 essential proteins from an initial group\nof 353 proteins. To improve clarity, we have revised the text to more explicitly describe the rationale and\noutcome of the filtering process. The sentence now reads: “Given Nudt21’s crucial role in self-renewal, we\nselected essential genes from the remaining 353 candidates based on the publicly available ‘essentialome’\ndataset DepMap, leading to 73 essential candidates for final analysis”(lines 384-386 in the text, page 9)\n7. Along these same lines, please consider changing the sentence: “Importantly, protein levels for the 73 candidate\nESSENTIAL genes decreased at day 4 even though…”\nWe appreciate the reviewer’s suggestion and have revised the sentence as follows: “Importantly, protein\nlevels for the 73 essential candidate genes decreased at day 4 even though associated RNA levels\nremained unchanged in Nudt21 KO cells (Figure 4d)”, (lines 415-417 of the text, page 10)\n8. The result that these 73 genes are enriched in multi-protein complexes is also quite compelling – but it is not clear\nif this observation is conserved in human. This seems easy enough to determine as the authors can just compare\nthese genes/gene structures to their human counterparts and query PADB. This would further strengthen their\ngeneral thesis.\nWe thank the reviewer for this helpful suggestion. To answer this question, we compared published APA\nsites (APA database) for the 73 candidates. We found that these genes contain significantly more PAS than\nexpected from randomized sampling distributions. Importantly, this enrichment is conserved between mouse\nand human orthologs, suggesting that the presence of multiple PAS is a general regulatory feature encoded\nwithin their 3′UTRs We have added this analysis to the text (lines 398-404, page 9) and as Extended Data\nFigure 6d and 6e.\nExtended Data Fig. 6:\nd and e: Number of PAS per gene for the 73 candidate genes compared to randomized groups of 73 genes in mouse (d) and human\n(e).\n9. The results in Figures 4/5 demonstrate the 3’UTR shortening of specific nucleoporin mRNA result in their loss of\nprotein expression despite no change in mRNA stability or translational competency. The authors posit that 3’UTR-\nmediated co-translational assembly as a possible explanation for this result. While I believe testing this mechanistic\nhypothesis is beyond the scope of this paper, it is not unreasonable to suggest to the authors that heterologous\nreporter experiments using luciferase-3’UTRs would be beneficial. In other words, if the protein encoded by the short,\nmedium, or long nucleoporin 3’UTR is no longer a nucleoporin and is not in need of cotranslational assembly, will its\nexpression/accumulation no longer be attenuated??\nWe thank the reviewer for this insightful question and experimental suggestion, which we have tried to\naddress in four different ways:\n(1) To determine whether the 3′UTR isoforms of Nup160 influence cell cycle progression, expression, or\nlocalization of any protein-coding genes, we performed transfection experiments using in vitro transcribed\n(IVT) RNA constructs encoding either an in-frame GFP-Nup160 ORF or a GFP ORF fused to short, medium,\nand long Nup160 3′UTR isoforms. We chose IVT RNA to avoid confounding effects from DNA-based\nexpression constructs, which might be subject to Nudt21-dependent APA regulation. Overexpression of long\nbut not short 3’UTR Nup160 isoforms linked to the GFP-Nup160 ORF led to cell cycle arrest in WT cells,\nconfirming strikingly different biological effects of distinct Nup160 3’UTR isoforms and suggesting that\nendogenous Nup160 3’UTR size needs to be tightly regulated endogenously to sustain cell division\n(Extended Data Fig. 10a). Nup160 3’UTR isoforms linked to GFP had only subtle effects on cell cycle\nprogression (Extended Data Fig. 10a). Moreover, GFP-Nup160 ORF constructs with the long 3’UTR led to\nreduced GFP levels than the short and medium 3’UTRs, supporting an isoform-dependent effect on protein\nlevels. Notably, reduced protein levels were also observed with the long 3’UTR GFP construct compared to\nthe medium and short 3’UTR GFP constructs, consistent with a coding sequence-independent effect on\nprotein levels (Extended Data Fig.10c-f). Together, these observations imply that Nup160 3′UTRs exert both\ncoding sequence-dependent and independent effects on posttranscriptional gene regulation, possibly by\ninfluencing translation efficiency and protein folding/stability in native cells. No significant changes in protein\nlocalization were observed between different 3’UTR isoforms (Extended Data Fig.10g).\n(2) To determine whether different Nup160 3’UTR isoforms are associated with different proteins, we\nperformed RNA immunoprecipitation (RNA-IP) using biotinylated versions of the three IVT Nup160 mRNA\nisoforms. This revealed that the long Nup160 3′UTR isoform preferentially binds to ribosomal subunits,\nsupporting the hypothesis that an extended 3′UTR facilitates ribosome recruitment or engagement, perhaps\nby enabling the co-translational assembly of Nup160 in the nuclear pore complex (Extended Data Fig.11).\nThese findings align with the previously proposed model of 3′UTR-mediated coordination between\ntranslation and protein complex assembly3 .\n(3) Overexpression of the long Nup160 3’UTR isoform was insufficient to rescue the cell cycle block caused\nby Nudt21 KO, suggesting that cell cycle-dependent expression is critical for proper Nup160 function and/or\nthat dysregulation of additional APA targets contributes to the cell cycle defect of Nup160 KO cells\n(Extended Data Fig. 10b).\n(4) Finally, we performed disome selective ribosome profiling (DiSP) in collaboration with Bernd Bukau’s lab\nto assess the global effect of Nudt21 loss on co-co-translationally assembled proteins. We discovered that\nnearly half of all co-co-translational protein assembly events detected in control cells were reduced in the\nabsence of Nudt21, and affected proteins were enriched for cell cycle regulators. These observations are\nconsistent with our cell cycle phenotype and support the notion that APA plays a critical role in co-co-\ntranslational assembly mechansims. Please note that nucleoporins did not score in the DiSP assay as they\ntypically assemble via a co-post-translational mechanism (REF Beck?).\nThese findings have been added to the text (lines 554-569, page 13).\nExtended Data Fig.10: Localization and expression efficiency of IVT eGFP-Nup160:\na and b: Percentage of GFP+ esophageal basal cells in S phase determined by EdU incorporation. Control cells (a) and Nudt21 KO\ncells (b) cells were transfected with GFP or GFP-Nup160 IVT mRNAs carrying short (S), medium (M), or long (L) Nup160 3′UTRs.\nc: Flow cytometry plots showing GFP expression in cells expressing the indicated IVT mRNA isoforms.\nc: Quantification of the percentage of GFP-positive cells expressing the indicated IVT mRNA isoforms.\nd: Percent of GFP expression in esophageal cells transfected with IVT mRNA isoforms.\ne and f: mean fluorescence intensity (MFI) of samples in (d)\ng: Representative fluorescence microscopy images of esophageal basal cells\nExtended Data Fig.11: The Long Nup160 3’UTR isoform interacts with ribosomes\na: Schematic illustrating RNA-IP/MS strategy. Equimolar amounts of IVT mRNAs were used per condition.\nb: Heatmap showing differential association proteins with the Nup160 3’UTR isoforms detected by mass spectrometry.\nd: Gene Ontology (GO) enrichment of protein complexes associated with Nup160 Long 3’UTR isoform, emphasizing ribosome- and\nribonucleoprotein-related complexes.\nData represent two independent experiments.\nExtended Data Fig. 12: Nudt21 KO disrupts co-co translation of cell cycle factors.\na: Rank-ordered DiSP co-co-assembly scores in control cells. 143/4,986 translated mRNAs meet the criterion for co-co translational\nassembly.\nb: Scatterplot of co-co-assembly score change (Nudt21 KO vs control). The orange quadrant indicates co-co assemblies impacted\nby Nudt21 KO.\nc: Dot plot showing all factors from the orange quadrant in (b), indicating whether they are Nudt21 targets based on PAS-seq\nanalysis.\nd: Gene Ontology (GO) enrichment of the 63 disrupted mRNAs in (b), highlighting cell cycle relevant terms.\ne: Heatmap of co-co assembly scores for representative nuclear-envelope–associated factors and their functions.\nData represent two independent experiments.\nWe thank this reviewer for their overall interest in our study and their recommendation to definitely publish\nour findings.\nSpecific points:\n1. The miRNA analysis does not make the paper stronger. It is only correlative and does not add real insights. Even if\nloss of miRNA regulation would cause slight upregulation of some of the factors shown in Fig. 3i, it is unclear whether\nthis has any relevance for the phenotype observed. In my opinion, the paper would benefit from leaving this part (Fig.\n3f-3l and Ext Data Fig. 4f-4g) out as it does not add anything to the proposed mechanism by which loss of Nudt21\ncauses death.\nWe thank the reviewer for their suggestions to streamline the paper. We’d like to clarify our reasoning for\nincluding the miRNA data and our preference for keeping these results in the manuscript. Please note that\nthese experiments were not intended to explain the cell cycle arrest caused by complete Nudt21 ablation,\nbut rather to explore the distinct phenotypes observed in Nudt21 hypomorph cells, which exhibit a\ndifferentiation defect but no cell cycle defect, versus Nudt21 KO cells, which undergo a profound cell cycle\narrest.\nOur characterization and comparison of the miRNA/differentiation and protein complex/cell cycle phenotypes\nin Nudt21 hypomorph and KO cells, respectively, support the fundamental conclusion that Nudt21 acts in a\ndose-dependent manner to regulate distinct cellular processes, and that these processes can be molecularly\nand functionally uncoupled. Our data may further help to explain conflicting observations in the literature\nrelated to Nudt21’s opposing roles in oncogenesis as well as other processes. As such, we believe that our\ndissection of miRNA regulation in the context of the differentiation phenotype adds value and contrast to the\nNudt21 KO-specific self-renewal/lethality phenotype, particularly in light of previous literature.\nHowever, to address this reviewer’s concern and further strengthen this part of the manuscript, we have\nconducted the following additional experiments:\n1. Functional evidence for specific miRNA interactions: We show that disruption of specific mRNA-\nmiRNA interactions (i.e. Jmjd1c-miR34a) with antagomirs delays the differentiation of WT stem\ncells akin to Nudt21 hypomorph cells, thus functionally connecting specific miRNAs to the Nudt21\nphenotype. These results are discussed on page 8, lines 342-351 and shown in Figure 3i (copied\nbelow) and Extended Data Fig. 4. Please also see our response to reviewer 2, question 5 and\nreviewer 1, question 2.\nFigure 3: Modulation of CFIm levels uncouples self-renewal from differentiation\ni: Relative mRNA and protein expression levels of genes following the model in (h), as well as expressed miRNAs predicted to target\nthese mRNAs/proteins in self-renewing esophageal basal stem cells. Cells were harvested for analysis after 4 days of ETOH or 4OHT\nor 4OHT+Dox treatment. Cut-offs: log2FC ≥1.3, and FDR ≤ 0.05.\n2. Network-level analysis: We performed a correlation analysis for specific competing endogenous\nRNA (ceRNA)/miRNA pairs in WT versus Nudt21 hypomorph cells, which revealed that Nudt21-\ndependent 3′UTR shortening disrupts the coordinated expression of miRNA-regulated gene\nnetworks, further strengthening the role of 3′UTR dynamics in differentiation. These results have\nbeen added to the text (lines 353-373, pages 8-9) and to Extended Data Figure 5. Please also see\nour response to reviewer 1, question 2.\n3. Manuscript title: Based on these new data, and supportive feedback from Reviewers 1 and 2, we\nbelieve the miRNA results are integral to the manuscript as they illustrate one crucial function of\nNudt21’s dual role in tissue homeostasis: the regulation of differentiation via miRNA/3′UTR\ninteractions. This result is contrasted by Nudt21’s other crucial function: the regulation of self-\nrenewal via modulating protein complex stability. To better emphasize this dual role of Nudt21 in\ntissue maintenance, we have revised the title to: “ Modulation of Nudt21 Levels Reveals Dose-\ndependent Roles of Alternative Polyadenylation in Tissue Regeneration”\n2. Nudt21 affects the nuclear pore.\nIt is currently unclear, what the authors think is the molecular mechanism by which Nudt21 loss affects the nuclear\npore. If Nup160, Nup107 and Seh1L are essential genes and they are knocked-down, it is not surprising that the cells\nshow decreases in cell cycle progression.\nWe agree that cell cycle defects caused by the knockdown of essential genes are not surprising when\nconsidered in isolation. However, our siRNA experiments (Extended Data Fig. 8d) clearly show that\nsuppression of Nudt21 reduces Nup160 protein levels, whereas suppression of Nup160 reduces Nup160,\nNup107, and Seh1l protein levels without perturbing Nudt21 levels, supporting the notion that Nudt21 acts\nupstream of Nup160. Consistent with this proposed epistatic relationship, suppression of either Nudt21 or\nNup160 resulted in a profound cell cycle arrest. Most importantly, we demonstrate that CRISPR-mediated\ndeletion of the alternative Nup160 3′UTR and RNAi-mediated silencing of the long Nup160 3′UTR isoform\nare sufficient to elicit a significant cell cycle defect (Fig. 5j and Extended Data Fig. 9n). These findings\nestablish a novel mechanistic link between Nudt21, a specific Nup160 APA isoform, and cell-cycle\nprogression, expanding our understanding of post-transcriptional regulation of essential protein complex\nsubunits.\nMoreover, the figure with the chromatin bridges in Ext Data Fig. 5j, k should be removed. If the authors think that\nformation of the chromatin bridges are causative, it needs to be demonstrated.\nPlease note that we do not state anywhere in the manuscript that the chromatin bridges we detected in\nNudt21 KO cells are causative for the cell cycle arrest. However, we emphasize the fact that DNA damage\nand genomic instability are well-established consequences of nuclear pore dysfunction, especially when\ncomponents of the Y-complex such as Nup133 are disrupted. Indeed, recent work from Martin Beck’s group\n(PMID: 40205196) confirmed this result. Thus, we prefer to keep these data in the manuscript as they\nestablish an important correlative link between Nudt21, nuclear pores and genomic stability. We have made\nsure to remove or down-tone any statements from the text that imply causality.\nIf they think that decreased Nup160 protein levels are causative for the phenotype, it is important to restore Nup160\nprotein level to investigate if this rescues some of the phenotypes seen in Nudt21 loss of function cells.\nWe thank the reviewer for this suggestion. We have attempted rescue experiments using expression\nconstructs for the short, medium, and long 3′UTR isoforms of Nup160 (Extended Data Fig. 10, see response\nto reviewer 2 question 9). However, none of the isoforms were able to rescue the cell cycle defect when\nintroduced into Nudt21 KO cells. Considering that thousands of target genes undergo 3’UTR shortening in\nNudt21 KO cells in addition to Nup160, we believe that overexpression of a single 3’UTR isoform may be\ninsufficient to exert a rescue. Moreover, our observation that Nup160 3’UTR isoform expression dynamically\nchanges with the cell cycle may also explain why constitutive (i.e. non cell cycle-dependent) expression of\nsingle 3’UTR isoforms fail to reverse nuclear pore and cell cycle defects (Extended Data Fig.9a-f).\nAdditionally, we explored whether 3′UTR-mediated regulation of cell cycle progression is unique to Nup160\nor part of a broader mechanism involving other target genes and protein complexes. To this end, we deleted\nthe 3′UTRs of additional candidate genes impacted by Nudt21 loss using a targeted CRISPR screen.\nIndeed, we identified several genes whose 3′UTR deletion causes similar defects in cell proliferation\ncompared to Nup160. These findings suggest that multiple 3′UTR-regulated genes likely contribute to the\nobserved cell cycle defect, and that rescuing the Nudt21 loss-of-function phenotype by manipulating a single\ntarget gene may be difficult if not impossible (Rebuttal Figure 1 below). Since this additional screen is part of\nan ongoing follow-up project, we prefer not to include it in this manuscript but are happy to share it with the\nreviewers.\nWdr12\nRbm28\nAlg14\nSap18\n1\n0\n−4 −3 −2 −1 0 1\nlog2FC\nRebuttal Figure 1: Complementary dgRNA screen\nQuantification of dgRNAs enrichment of selected targets based on their 3’UTR change upon Nudt21 KO. dgRNA enrichment ratios\nwere derived by comparing dgRNA frequencies in Cas9-on (+DOX) vs Cas9-off (-DOX) conditions.\nOr, if the authors think that the longest 3’UTR isoform of Nup160 is responsible for protein complex assembly with\n)RDF(01gol−\n+DOX vs −DOX Focused dgRNA library\nother proteins in the nuclear pore, then, direct evidence needs to be shown. At the current state of the manuscript,\nlots of data are presented, but the overall message of this part is not clear.\nWe appreciate the reviewer’s comment regarding the mechanistic role of the long 3′UTR isoform of Nup160\nin nuclear pore complex assembly.\nMechanistically, we propose a model whereby the long 3′UTR of Nup160 contributes to nuclear pore\ncomplex assembly or stability by facilitating the co-translational interaction of subunits. In support of this\nnotion, we found that the long 3′UTR isoform interacts more strongly with ribosomal subunits using pull-\ndown experiments with in vitro transcribed (IVT) and biotinylated Nup160 transcripts (Extended Data Fig.11,\nsee response to reviewer 2, question 9). Consistent with this model, 3′UTRs have recently been suggested\nto help orchestrate spatial translation, folding, and complex assembly3,4. To experimentally support this\nmodel, we performed DiS, which captures co-co-translational protein assembly events. Co-co-translational\nassembly is defined as a translational process in which a nascent polypeptide chain interacts with another\nnascent polypeptide chain, in contrast to co-post-translational assembly, where a nascent polypeptide chain\ninteracts with an already folded protein. Although Nup160 did not score in the DiSP assay, we found that\nalmost half of all co-co-translationally assembled proteins are impacted by Nudt21 KO and that most of\nthese factors are involved in cell cycle processes, supporting a hitherto unknown function of Nudt21 in the\nregulation of co-co-translational assembly processes (Extended Data Fig. 12, lines 570-590 of the text, page\n13, see response to reviewer 2 question 9). To address the question of how the 3’UTR of Nup160 influences\nnuclear pore assembly, future experiments outside the scope of the current manuscript (e.g. co-post\ntranslational assembly assays) are required to definitively prove this model. Of relevance, recent evidence\nsuggests that conserved 3′UTRs can modulate the biogenesis of intrinsically disordered region (IDR)-\ncontaining proteins by acting as RNA-based chaperones5 . In this framework, 3′UTRs engage in multivalent\ninteractions with IDRs during translation, influencing oligomerization states and folding kinetics in\ncondensate-like environments. It is conceivable that such a mechanism also applies to Nup160, which\ncontains extended domains with structural flexibility, and may help explain how its long 3′UTR contributes to\nthe assembly of stable nuclear pore complexes.\nWhile we acknowledge that additional experiments will be required to distinguish between these\nmechanisms, our data and models provide a useful conceptual framework for follow-up experiments (e.g.,\ndirect co-post translational assembly assays or tests for 3′UTR–IDR interactions).\nWe are discussing this alternative hypothesis in the manuscript on page 15, lines 668-684 of the text.\n• 3. The data in Fig. 4g showed upregulation of 26S proteasome complexes in the KO. As an alternative\nexplanation, could this contribute to the observed protein destabilization, and therefore not all observed\ndecreases in protein levels are direct consequences of APA-dependent protein complex formation? Importantly,\nmost essential genes, not just genes regulated by Nudt21 through APA, are downregulated in the KO, which\nfurther raise the question of the direct causal relationship between its role in APA and the phenotype.\nIn the critical Fig./Extended Fig 5, which should help to establish causal relationship between APA and the\nphenotype, there are some problems: (1) Regarding phenotype in mRNA export, images in Nup160 long isoform\ndeletion is inconsistent with what is shown in the quantification. I think a better way of presenting the result is to\nplot the fraction of mRNA exported/cell, rather than the absolute number. (2) The siRNA targeting the middle\nisoform is not isoform-specific and leads to equal decrease in the mRNA level of the longest isoform and of\nNup160 protein levels.\nWe appreciate the reviewer’s question about the potential contribution of proteasome-dependent\ndysregulation of protein subunits not directly regulated by APA to the Nudt21 KO phenotype. In fact, we\ndeem it likely that such a mechanism contributes to global protein dysregulation in Nudt21 KO cells following\nthe protein downregulation of direct APA targets. In support of this idea, GO analysis of upregulated proteins\nin Nudt21 KO cells points to “Regulation of protein-containing complex assembly”, “Autophagy”, and “Protein\nfolding”, suggesting that global protein complex formation is indeed de-regulated and “orphan” proteins\neither aggregate and/or become degraded subsequent to the downregulation of direct APA targets. In further\nagreement with this notion, we observed upregulation of certain chaperones involved in protein folding,\nconsistent with a proteotoxic stress response elicited by the accumulation of unfolded proteins.\nTo test whether disrupted protein complex stoichiometry and resultant proteotoxic stress are functionally\ninvolved in the Nudt21 KO phenotype, we treated Nudt21 KO cells with ISRIB, an inhibitor of the integrated\nstress response (ISR). Indeed, ISRIB treatment delayed, but did not fully rescue, the Nudt21 KO-associated\ncell cycle arrest, suggesting that proteostatic defects contribute to but are not the sole driver of the Nudt21\nKO phenotype (Extended Data Fig. 7e). We have added this result to the revised manuscript and now\ndiscuss this mechanism as a likely contributor to the Nudt21 KO-specific phenotype (page 10, lines 424-440\nof the text,Extended Data Fig. 7). Please also see our response to reviewer 1, question 1.\nIt’s important to note that our model does not require all downregulated, essential proteins to be direct APA\ntargets. Downregulation of individual protein subunits directly regulated by APA is expected to be sufficient to\ntrigger the subsequent degradation of the entire protein complex, including subunits that are non-APA\ntargets yet essential. Thus, the observed involvement of the ISR downstream of Nudt21 KO makes\nbiological sense.\nWith regards to the quantification of the mRNA export phenotype, we have re-plotted the mRNA export data\nas the fraction of mRNAs exported per cell, normalized to total nuclear and cytoplasmic signal, which\nprovides a more accurate reflection of export efficiency and confirms a robust mRNA export defect in Nudt21\nKO cells. The revised figure and quantification are now included in Figure 5g and p.\nFigure 5: Nuclear pore integrity & function are regulated by APA via CFIm\nf: Poly(A) mRNA FISH assay to assess mRNA export efficiency in Nudt21 KO esophageal stem cells (day 4).\ng: Quantification of cytoplasmic poly(A) foci in (f).\no: Immunofluorescence staining with mAb414 antibody to assess the effect of Nup160 3’UTR deletion on nuclear pore signal.\np: Quantification of nuclear pore signal in (o).\nRegarding siRNA specificity, we fully agree with the reviewer that single siRNAs cannot deplete each of the\nNup160 isoforms and this experiment simply served as a “positive” control. However, in combination with the\ndistal-specific siRNAs, which target exclusively the long 3′UTR isoform, as well as the CRISPR-based\ndeletion of the long 3’UTR isoform, we feel confident that the long Nup160 isoform plays a key role in\nnuclear pore and cell cycle-associated defects.\nIn summary, we do not wish to claim that APA alone explains every aspect of the Nudt21 KO phenotype.\nRather, our data are consistent with the interpretation that APA-driven dysregulation of protein complex\nsubunits significantly contributes to the phenotype, as exemplified by the nuclear pore complex, and that\nresultant proteostatic stress and the Integrated Stress Response likely play an important downstream role in\nthis phenotype. We have revised the manuscript to present this more nuanced model and to incorporate the\nsuggested improvements in data presentation and interpretation.\n4. A strength of the paper is the genome-wide and proteome-wide analyses in a time course of Nudt21 depletion. As\nthe authors seem to have problems of pinpointing to the mechanism responsible for the observed phenotype, it is\nessential to provide the entire dataset for other researchers to use (as supplementary table). This should include all\nthe data on APA, all the mass spectrometry data (not just the data on the essential differentially expressed proteins).\nAlso, the data on the hypomorph should be included in this table. If this table is presented well (with p-values and\nfold-changes), it would be a useful resource for the community to test alternative hypotheses on the mechanism by\nwhich Nudt21 has such strong effects on self-renewal and differentiation.\nWe thank the reviewer for highlighting the strength of our genome-wide and proteome-wide analyses. We\nfully concur with this suggestion and have provided the complete datasets as supplementary tables. Each\ntable includes p-values and fold-change values."}, {"round": 2, "reviewer_comments": {"reviewer_2": "I was already pleased with the quality and rigor of the manuscript upon first submission and this revision only further\nstrengthens that position. I commend the authors on their efforts to address my comments and I am now in complete support\nof publication.", "reviewer_3": "The authors have revised their manuscript to my full satisfaction.\nPlease accept it.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": ""}]

Decision letter