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# Ablation of ZC3H11A causes early embryonic lethality and dysregulation of metabolic processes

Shady Younis, Alice Jouneau, Mårten Larsson, Jean-François Oudin, Vincent Brochard, Leif Andersson

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Shady Younis, Alice Jouneau, Mårten Larsson, Jean-François Oudin, Vincent Brochard, et al. Ablation of ZC3H11A causes early embryonic lethality and dysregulation of metabolic processes. 2023. [hal-03814143](https://hal.inrae.fr/hal-03814143)

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Preprint submitted on 6 Jun 2023

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1 Ablation of ZC3H11A causes early embryonic lethality and dysregulation of
2 metabolic processes

3

4 Shady Younis¹,²,*, Alice Jouneau³, Mårten Larsson¹, Jean-François Oudin³, Vincent Brochard³ and Leif Andersson¹,⁴,*

6

7 ¹Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University,

8 SE-751 23 Uppsala, Sweden. ²Division of Immunology and Rheumatology, Stanford University, Stanford,

9 CA 94305, USA. ³Université Paris-Saclay, UVSQ, INRAE, BREED, 78350 Jouy-en-Josas, France.

10 ⁴Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843,
11 USA.

12

13 * To whom correspondence should be addressed.

14 Email: syounis@stanford.edu or leif.andersson@imbim.uu.se.
15 Abstract

16 ZC3H11A is a stress-induced mRNA binding protein required for efficient growth of nuclear-
17 replicating viruses, while being dispensable for the viability of cultured human cells. The cellular
18 functions of ZC3H11A during embryo development are unknown. Here we report the generation
19 and phenotypic characterization of *Zc3h11a* knock-out mice. Heterozygous null *Zc3h11a* mice
20 were born at the expected frequency without distinguishable phenotypic differences compared
21 with wild-type. In contrast, homozygous null *Zc3h11a* mice were missing, indicating that *Zc3h11a*
22 is crucial for embryonic viability and survival. *Zc3h11a*<sup>-/-</sup> embryos were detected at the expected
23 Mendelian ratios up to late preimplantation stage (E4.5). However, phenotypic characterization at
24 E6.5 revealed degeneration of *Zc3h11a*<sup>-/-</sup> embryos, indicating developmental defects around the
25 time of implantation. Transcriptomic analyses documented a dysregulation of glycolysis and fatty
26 acid metabolic pathways in *Zc3h11a*<sup>-/-</sup> embryos at E4.5. Proteomic analysis indicated a tight
27 interaction between ZC3H11A and mRNA-export proteins in embryonic stem cells. Furthermore,
28 CLIP-seq analysis demonstrated that ZC3H11A binds a subset of mRNA transcripts that are
29 critical for metabolic regulation of embryonic cells. Altogether, the results show that ZC3H11A is
30 participating in export and post-transcriptional regulation of selected mRNA transcripts required
31 to maintain metabolic processes in embryonic cells. While ZC3H11A is essential for the viability
32 of the early mouse embryo, inactivation of *Zc3h11a* expression in adult tissues using a conditional
33 knock-out did not lead to obvious phenotypic defects.
## 34 Introduction

The zinc finger CCCH domain-containing protein 11A (ZC3H11A) is a stress-induced mRNA-binding protein that is required for the efficient growth of several human nuclear replicating viruses, including human immunodeficiency virus (HIV-1), influenza A virus (IAV), human adenovirus (HAdV) and herpes simplex virus 1 (HSV-1) [1]. Proteomic studies on human cells have indicated that ZC3H11A is a component of the transcription-export (TREX) complex [2]. Functional studies indicated that ZC3H11A selectively export newly transcribed viral mRNAs to the cytoplasm during virus infection [1, 3]. Thereby, inactivation of ZC3H11A in human cells impaired the export of a subset of viral mRNA transcripts and resulted in a dramatic reduction in virus growth [1]. These important functions of ZC3H11A in the growth cycle of several human viruses makes ZC3H11A a potential target for development of an anti-viral therapy. The aim of the present study was to develop an animal model to study the molecular functions of ZC3H11A in prenatal and postnatal development.

The TREX complex serves a key function in nuclear mRNA export and consists of multiple conserved core subunits including ALYREF (RNA binding adaptor of TREX), UAP56 (DEAD-box type RNA helicase) and a stable subcomplex called THO, which in turn consists of at least six subunits [4, 5]. Proteomic studies using human cells have indicated that ZC3H11A is an auxiliary component of the TREX complex, but did not consider it as a core subunit of the TREX complex [6, 7]. THO proteins are conserved from yeast to human and play pivotal roles during embryo development, cell differentiation and cellular response to stimuli [8, 9]. It has been reported that the disruption of THO proteins, such as THOC1, THOC2 or THOC5, leads to early embryonic lethality [9–11]. The TREX complex controls the mRNA export in a selective manner, where individual TREX components appear to be required for export of distinct subsets of mRNAs [12].
For instance, THOC2 or THOC5 are required for the export of mRNAs essential for pluripotency such as Nanog, Sox2 and Klf4 in mouse embryonic stem cells [9]. Despite several reports characterizing the role of THO proteins during embryogenesis, the cellular function of ZC3H11A during embryo development is unknown.

In the current study, we established Zc3h11a knock-out (KO) mouse models to study the effect of Zc3h11a loss of function on embryo development. Our results identify ZC3H11A as a fundamental protein required for early embryo growth. Disruption of ZC3H11A is homozygous lethal and leads to complete failure of embryo development and survival. Using proteomic and RNA-seq analyses, we show that the ZC3H11A protein interacts with TREX complex core proteins in mouse embryonic stem cells. ZC3H11A is apparently an auxiliary factor participating in export and post-transcription coordination of selected mRNA transcripts required to maintain the metabolic processes in embryonic cells. Interestingly, Zc3h11a inactivation in adult mouse tissues using an inducible mouse model showed that the ZC3H11A protein is dispensable for postnatal tissue growth.

## 71
# Results

## 72
## 73
### Zc3h11a inactivation in mice is lethal in the homozygous condition

Zc3h11a is located on chromosome 1 in both human and mouse genomes and harbors the coding sequence of another gene encoding the DNA-binding zinc-finger protein ZBED6 [13–18] (Figure 1A). We used two strategies to target the Zc3h11a coding exons without affecting Zbed6. The first Zc3h11a mouse model was developed by targeting exon 3 using the CRISPR/cas9 system with two guide RNAs flanking the targeted sequences. This resulted in both a deletion of 567 bp including the entire exon 3 and a frameshift (Figure 1B). The second mouse model was developed by inserting loxP sites flanking the coding sequence of exon 2 using homologous recombination
to visualize the ZC3H11A protein and the nuclear speckles marker SRSF2 (SC35) for expression profiling in mouse 2-cell and blastocyst stages. The IF analysis indicated that ZC3H11A was expressed at a detectable level as early as the 2-cell stage, with clear nuclear localization (Figure 3A, top panel). The z-stack imaging of the blastocysts showed that ZC3H11A was expressed in trophectoderm (Figure 3A, middle panel) as well as in inner cell mass (ICM) (Figure 3A, bottom panel). The localization pattern of ZC3H11A in ICM was overlapping with the nuclear speckles as indicated using the anti-SC35 antibody (Figure 3A and B). This subcellular localization of ZC3H11A in mouse embryonic cells is similar to the ZC3H11A localization in human cell lines [1]. Re-analyzing single cell RNA-seq data from Deng et. al. [19] revealed that Zc3h11a mRNA is highly expressed in mouse embryos as early as the zygotic stage, indicating maternal contribution (Figure 3C).

## Disrupted metabolic pathways in the Zc3h11a<sup>-/-</sup> embryos

The degeneration of Zc3h11a<sup>-/-</sup> embryos during early embryo development (E6.5) encouraged us to perform whole transcriptome analysis of stage E4.5 embryos to reveal the dysregulated pathways that led to the degeneration of Zc3h11a<sup>-/-</sup> embryos at E6.5. We collected embryos from Zc3h11a<sup>+/-</sup> X Zc3h11a<sup>+/-</sup> matings and extracted the RNA from the embryonic part for sequencing (Figure 4A, left). Principle component analysis (PCA) of RNA-seq data showed that Het (Zc3h11a<sup>+/-</sup>) and WT (Zc3h11a<sup>+/+</sup>) embryos clustered together and apart from the KO (Zc3h11a<sup>-/-</sup>) embryos (Figure 4A). This result is in agreement with the observed morphological similarity between WT and Het embryos (Figure 2B). Furthermore, the differential expression (DE) analysis between WT and Het did not detect any significant DE genes with FDR <0.05. Therefore, we performed the DE analysis between KO embryos vs. WT and Het embryos that revealed 660 DE
genes (FDR <0.05) out of ~11,000 expressed genes (Table S1). Among these DE genes, 419 were up-regulated and 241 were down-regulated in KO embryos (FDR <0.05). Next, we performed a gene set enrichment analysis (GSEA) using the DE genes in KO embryos in order to further explore the function of ZC3H11A. The GSEA of ranked DE genes in KO embryos using the hallmark gene sets revealed a significant negative enrichment (FDR <0.05) of genes involved in glycolysis, fatty acid metabolism pathways and epithelial-mesenchymal transition (EMT) processes (Figures 4B-4D). The heatmaps present the expression of the subset of genes that contributed the most to the indicated pathway enrichment among significantly down-regulated genes in KO embryos (Figures 4B-4D). Among the key down-regulated genes, contributing to the significant GSEA result, are lactate dehydrogenase A (*Ldha*), which has an essential role in glycolysis, and disruption of *Ldha* causes congenital disorders of carbohydrate metabolism [20, 21]; enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (*Ehhadh*), which is involved in fatty acid beta-oxidation using acyl-CoA oxidase [22, 23]; and dickkopf WNT signaling pathway inhibitor 1 (*Dkk1*), which is involved in several processes including cell fate determination and cell differentiation processes during embryogenesis [24]. On the other hand, the positively enriched gene sets among up-regulated genes in the KO mice included genes in the P53 pathway and autophagy process-related genes (Figures 5A, 5B and S1). This includes the up-regulation of autophagy related 12 (*Atg12*) and microtubule-associated proteins 1A/1B light chain 3A (*Map1lc3a*) genes (Figure 5E). MAP1LC3A is known as LC3A protein and is required for autophagosome formation [25].

To get further insight on which cell type in blastocysts was most affected by *Zc3h11a* inactivation, we explored the expression profile of the DE genes in *Zc3h11a*⁻/⁻ embryos in embryonic lineages (ICM and epiblast) and trophectoderm (TE). Using previously published
datasets of mouse gene expression (GSE76505 [26]), the ICM/TE ratio of expression was computed for genes down-regulated and up-regulated in the KO embryos (FDR ≤0.05, fold change ≥2). We also explored the expression of DE genes at earlier stages using gene expression dataset (E-MTAB-2950) [27]. This showed that down-regulated genes in Zc3h11a⁻/⁻ embryos are primarily expressed in the ICM/early epiblast rather than trophectoderm (Figure 5C), while the expression of the up-regulated genes is nearly equally present in ICM/early epiblast and TE (Figure S2). The down-regulated genes in Zc3h11a⁻/⁻ embryos with high expression in the ICM include: *Ldha*, teratocarcinoma-derived growth factor (*Tdgf1*, *Cripto*), growth differentiation factor 3 (*Gfd3*), phosphofructokinase (*Pfkp*) (Figure 5D). GDF3 is an analog of NODAL and uses TDGF1 as co-factor [28]. Both *Ldha*, *Pfkp*, *Pfkm* and *Pdk2* are down-regulated in Zc3h11a⁻/⁻ embryos and are involved in glycolysis and lactate production, as indicated in the GSEA (Figure 4). At peri-implantation stages, there is a major metabolic switch from oxidative phosphorylation to anaerobic glycolysis, with increased lactate production [29, 30]. *Cripto/Tdgf1* has been reported as an essential factor regulating this metabolic switch [31]. That explains the GSEA results that show that the down-regulated pathways mostly concern metabolic regulation processes. Altogether, this strongly suggests that the primary consequence of ZC3H11A deficiency is in the ICM, due to perturbed metabolic regulation. The enrichment of genes associated with autophagy and apoptosis-related pathways (Figure 5A-5B) among the up-regulated genes in Zc3h11a⁻/⁻ embryos could be secondary effect caused by the metabolic stress encountered by the ICM cells [32, 33].

## ZC3H11A is associated with the RNA export machinery in embryonic stem cells

In human somatic cells, ZC3H11A has been recently characterized as an RNA-export protein that functions through its interaction with TREX complex proteins [1]. In order to identify its
175 interacting partners in embryonic cells and to investigate if ZC3H11A maintains its association
176 with the TREX complex in mouse embryonic stem cells (mESCs), we performed co-
177 immunoprecipitations (co-IPs) using anti-ZC3H11A, anti-THOC2 and anti-IgG antibodies
178 followed by mass spectrometry (MS) analyses (Figure 6A). Statistical analyses of detected MS
179 intensities from the biological replicates (n=4) revealed a number of proteins with statistically
180 significant interaction with ZC3H11A and THOC2 (Figure 6B). Proteins belonging to the TREX
181 complex and RNA export machinery are highlighted in bold. The log-fold change in protein
182 intensities in the ZC3H11A co-IP relative to the IgG co-IP is presented along with the adjusted P-
183 values (Figure 6C). The interaction between ZC3H11A and THOC2 was validated by a reciprocal
184 co-IP and western blot using mESCs (Figure 6D). The majority of the significant partners
185 interacting with ZC3H11A are part of the TREX complex and also showed significant enrichment
186 in the THOC2 co-IP, including THOC5, THOC7 (Figure 6E), THOC1 and THOC6 (Figure 6C).
187 ZC3H11A also interacts with other RNA-binding proteins that are required for RNA maturation,
188 such as polyadenylate-binding nuclear protein 1 (PABPN1) [34]; FYTTD1, which acts as an
189 adaptor for RNA helicase UAP56 [35]; and the RNA export adaptor ALYREF/THOC4 [36]
190 (Figure 6D and F). Notably, almost half of the ZC3H11A partners detected by co-IP were also
191 found in the THOC2 co-IP (Figure S3). These data indicate that ZC3H11A is an essential
192 component of the TREX complex that is known to play pivotal roles during embryogenesis and
193 for maintaining pluripotency of ESCs [9, 10]. Furthermore, the proteomic analysis identified
194 additional interacting partners, independent of the TREX complex, such as the RNA-binding
195 protein DDX18; the PRC2 components SUZ12 and JARID2; and the two zinc finger proteins
196 ZNF638 and ZFP57 (Figure 6G and S3B). DDX18 is an RNA binding protein that plays a crucial
197 role in pluripotency and self-renewal of embryonic stem cells [37].
198

199

ZC3H11A selectively binds mRNA transcripts in mESCs

200 Previous studies using human somatic cells indicated that ZC3H11A is an RNA-binding protein
201 that selectively binds subsets of mRNA upon stress or viral infection [1]. To study the RNA-
202 binding properties of ZC3H11A in embryonic cells, we performed UV-crosslinking of mESCs
203 followed by ZC3H11A immunoprecipitation (CLIP) and RNaseI treatment to isolate the RNA
204 protected by ZC3H11A. We used two anti-ZC3H11A antibodies to minimize any artifact caused
205 by antibodies, and anti-ALYREF and anti-IgG as positive and negative controls, respectively.
206 High-throughput sequencing of the RNA isolated by CLIP (CLIP-seq) revealed an almost
207 exclusive interaction between ZC3H11A and protein-coding mRNAs in mESCs (Figure S4A),
208 with a preference to bind 3'UTRs over the 5'UTRs (Figure 7A). The analysis of ZC3H11A CLIP-
209 seq peaks from the two ZC3H11A antibodies revealed a significant enrichment of short purine-
210 rich motifs (Figure 7B, top panel). Moreover, ZC3H11A exhibited strong binding to the
211 paraspeckle *Neat1* transcript (Figure S4B), similar to what has been observed in human somatic
212 cells [1]. Comparing the CLIP-seq ZC3H11A mRNA targets with genes that were significantly
213 down-regulated in RNA-seq data, we identified subsets of genes as putative direct targets of
214 ZC3H11A in mESCs (Figure 7B, bottom panel). The gene ontology analysis of these genes
215 suggested that they are involved in germ cell development and metabolic processes (Figure 7C).
216 These 29 genes were dramatically down-regulated in *Zc3h11a*-/- embryos (Figure 7D) and are
217 involved in cellular processes vital for embryonic development [38-41]. Putative direct targets
218 included the *Tdgfl*, nucleoporin 85 (*Nup85*), proliferation-associated protein 2G4 (*Pa2g4*) and gap
219 junction protein beta 3 (*Gjb3*) genes. The CLIP-seq analysis detected ZC3H11A binding sites at
220 the 3'UTR of these genes that were either overlapped with ALYREF binding sites (for *Tdgfl* and
221 *Pa2g4*) or distinct from them in *Nup85* (Figure 7E). These results suggest a crucial role of
222 ZC3H11A in post-transcriptional processing and mRNA export of key genes in embryonic cells.

223

224 ZC3H11A is required for *in vitro* derivation of ESCs.

225 To further understand the role of ZC3H11A in the peri-implantation development and especially
226 its role in the pluripotent epiblast, twenty-five E3.5 blastocysts were recovered from matings
227 between heterozygous mice, and cultured *in vitro*. From these, 14 ESC lines were obtained but
228 none were homozygous KO ($X^2=4.7, d.f.=1; P<0.05$). This suggests that ZC3H11A is required for
229 establishing ESC *in vitro*.

230

231 Mice with postnatal Zc3h11a-ablation are healthy and viable

232 We developed an inducible *Zc3h11a*-KO model to assess the effect of *Zc3h11a* ablation
233 postnatally. *Loxp-Zc3h11a* mice were crossed with mice containing fusion of a mutated estrogen
234 receptor T2 and Cre recombinase (Cre-ER), allowing temporal control of floxed gene deletion
235 upon tamoxifen induction *in vivo* [42]. We generated a strain that is homozygous *Zc3h11a-loxp*
236 (*Zc3<sup>loxP/loxP</sup>*) with one copy of Cre-ER (*CRE.ER<sup>+</sup> Zc3<sup>loxP/loxP</sup>*) and crossed it with the original strain
237 (*Zc3<sup>loxP/loxP</sup>*) lacking Cre-ER. The offspring were injected with tamoxifen at week 3-4 after birth
238 (Figure 8A). Genotyping of the tamoxifen-injected mice at week 6 using genomic DNA from tail
239 biopsies revealed a balanced ratio between WT and induced KO (iKO) due to the presence/absence
240 of Cre-ER (Figure 8B). By injecting *CRE.ER<sup>+</sup> Zc3<sup>loxP/loxP</sup>* mice with tamoxifen postnatally we
241 succeeded in achieving >90% reduction of *Zc3h11a* expression in multiple adult tissues including
242 bone marrow, liver and spleen (Figure 8C and D). The examination of tamoxifen-injected mice
243 were carried out at week 12 and involved histology staining of multiple organs including stomach,
244 pancreas, small and large intestine tissues. The histology phenotyping did not exhibit obvious
245 defects between the floxed (WT) and iKO adult mice (Figure 8E and S5). Furthermore, the
246 measurement of body weight, dissected kidney and spleen tissues from WT and inducible ZC3-
247 KO adult mice did not show significant differences (Figure 8F).

248

249 **Discussion**

250 ZC3H11A is important for the growth of nuclear replicating viruses, where viruses take advantage
251 of the ZC3H11A protein to facilitate the export of their mRNA transcripts into cytoplasm.
252 Thereby, ZC3H11A is considered a possible target for development of anti-viral therapy. Hence,
253 we developed ZC3H11A mouse models to study its physiological functions across developmental
254 stages. The current study reports that ZC3H11A is an essential protein required for the viability of
255 mouse embryos. Loss of function of ZC3H11A leads to developmental defects and embryo
256 degeneration at peri-implantation stages associated with dysregulation of metabolic pathways such
257 as glycolysis and fatty acid metabolic processes. Interestingly, the defects mainly originate from
258 the epiblast, as most of the down-regulated genes are expressed predominantly in this lineage.
259 Moreover, even though ZC3H11A is expressed in all cells of the blastocyst, *Tdgf1*, one of its key
260 down-regulated target genes is expressed specifically in the epiblast cells [31]. *TDGF1* (also called
261 *Cripto*) is a membrane-bound protein, co-receptor for NODAL/GDF3 [43]. *TDGF1* and NODAL
262 signaling play important roles during specification of the early lineages and maintenance of the
263 pluripotent epiblast at early post-implantation stages [43]. Interestingly it also controls the
264 metabolic switch occurring at the time of implantation in the mouse, when cells transit from a
265 OXPHOS based metabolism to a glycolytic one [29, 31, 44]. Our CLIP-seq analysis detected two
266 strong peaks for ZC3H11A binding at the 3' end of the *Tdgf1* mRNA in mESCs (Figure 7E).
Furthermore, ZC3H11A binds the 3' end of *Nup85* and *Pa2g4* mRNA transcripts (Figure 7E).

Both *Nup85* and *Pa2g4* were down-regulated in the KO embryos and play crucial roles in embryonic development [38-41]. For instance, NUP85 is a core component of the nuclear pore complex (NPC) proteins and is required for mRNA export and maintenance and assembly of the NPC [40, 41, 45]. Loss of function studies showed that inactivation of the NPC proteins in mouse models resulted in early embryonic lethality [46-48]. Recent phenotypic characterization of the *Nup85* knock-out mouse model from the International Mouse Phenotyping Consortium (www mousephenotype org, accessed 22 August 2022) [49] has indicated complete preweaning lethality of *Nup85*⁻/⁻ mice. Furthermore, the ErbB3 binding protein-1 gene (*Ebp1/ Pa2g4*) is implicated in regulating the proliferation and differentiation during developmental stages. The *Pa2g4* knock-out mice exhibited growth retardation and were 30% smaller than wild-type mice [50]. A recent study has reported more severe phenotypes in *Pa2g4*-deficient mice with death between E13.5 and 15.5, massive apoptosis, and cessation of cell proliferation [38]. These putative ZC3H11A targets identified by CLIP-seq are known to be critical for embryonic viability and implicated in diverse cellular functions, disruption of their expression leads to embryonic degeneration.

Another key down-regulated gene in KO embryos is *Ldha*, the enzyme that controls the level of anaerobic glycolysis by catalyzing the transformation of pyruvate into lactate. Hence, in KO embryos, the establishment of a more anaerobic glycolysis is impaired, which compromises survival when the environment becomes more hypoxic as embryos implant. Upregulation of autophagy as observed in KO embryos can be viewed as reaction to a suboptimal metabolic environment [33]. Although KO embryos can survive up to E6.5, they have already undergone a process of degeneration, as suggested by the upregulation of P53 mediated apoptotic pathway
already at E4.5. The transcriptomic analysis also indicated a significant dysregulation in the EMT
process (Figure 4C). The EMT process is fundamental for embryo development and takes place
during implantation of the embryo into the uterus and during early gastrulation, where embryo is
transformed from a single layer to three germ layers. Defects in EMT and subsequently in
gastrulation usually lead to a failure in embryonic development [51, 52].

The ZC3H11A protein exhibited strong interactions with members of the RNA-export
machinery in ESCs and the top interacting partners with ZC3H11A are members of the TREX
complex, including THO proteins (Figure 6). The enrichment analysis of interacting partners with
ZC3H11A showed significant enrichment of proteins involved in metabolism of RNA, mRNA 3'-
end processing and transport of mature transcript to cytoplasm (Figure S3A). These proteomics
results are in agreement with the analysis of the CLIP-seq of ZC3H11A in mESCs that revealed
preferential binding at 3'UTRs over the 5'UTRs of target transcripts (Figure 7A). It also supports
the model of action that ZC3H11A interacts with TREX-complex proteins and contribute to
efficient mRNA maturation and export of the target transcripts. In agreement with this model,
several studies have described the pivotal roles of the TREX-complex in the embryonic
development [9–11]. THO proteins such as THOC1, THOC2 and THOC5 play essential roles
during early development but in a different way than ZC3H11A, as their depletion affects
pluripotency establishment and maintenance [9, 10]. In contrast, ZC3H11A depletion does not
directly affect pluripotency maintenance. The fact that Zc3h11a<sup>-/-</sup> blastocysts did not give rise to
ESC lines in the present study may be due to the metabolic impairment rather than a defect in
pluripotency maintenance, as they all form outgrowth, in contrast to Thoc1<sup>-/-</sup> embryos [10].

Our results provide evidence that ZC3H11A is required for the post-transcriptional
regulation of genes that are crucial for the embryonic cell. In contrast to the severe phenotypes in
Zc3h11a germline KO embryos, Zc3h11a inactivation in the adult tissues did not cause obvious defects. The phenotypic characterization of the inducible ZC3-KO adult mice indicated a dispensable role for ZC3H11A in adult tissues and a single surviving Zc3h11a⁻/⁻ female showed no pathological conditions, were fertile and gave birth to 10 progeny from three litters. Furthermore, complete inactivation of Zc3h11a in human and mouse cell lines did not lead to significant effects on cell growth or viability [1, 3].

319

320

## 321 Methods

### 322 Animal models

All mice were group-housed with free access to food and water in the pathogen-free facilities of Uppsala University and INRAE. All procedures described in this study were approved by the Uppsala Ethical Committee on Animal Research (#17346/2017), following the rules and regulations of the Swedish Animal Welfare Agency, and were in compliance with the European Communities Council Directive of 22 September 2010 (2010/63/EU). All efforts were made to minimize animal suffering and to reduce the number of animals used. The loxP Zc3h11a mouse model was generated by homologous recombination in mouse C57BL/6 ES cells (Cyagen, USA). The PGK-Cre mice expressing the Cre recombinase in the germ line [53] was obtained as gift from Klas Kullander's lab (Uppsala University). The CRISPR/cas9 Zc3h11a mouse model was purchased from the Mutant Mouse Resource & Research Centers (MMRRC, USA, Strain No: 043457-UCD). For inducible knock-out model, the mice containing fusion of a mutated estrogen receptor T2 and Cre recombinase (Cre-ER) was ordered from The Jackson Laboratory (USA, Strain No: 008463). Mice were genotyped (Tables S2) based on tail biopsies.
336

337 **Collection of mouse embryos**

338 The *Zc3h11a* heterozygous males and females were mated and the following day, the presence of
339 a vaginal plug was recorded. To determine the time of developmental lethality, females were
340 sacrificed at E6.5 and embryos dissected out from the decidual swellings. Their morphology was
341 recorded and each of them was then processed for genotyping. Samples for RNA-sequencing were
342 collected at peri-implantation stage (E4). These embryos were bisected using glass needles and
343 both parts were individually snap-frozen. The abembryonic part (mural TE) was used for
344 genotyping and the embryonic part (ICM and polar TE) for subsequent RNA extraction.
345

346

RNA sequencing

347 The collected embryonic ICM and polar TE (as described above) were used for RNA-seq library
348 preparation using the SMART-Seq HT Kit (Takara Bio USA, Inc.) following the manufacturer's
349 instructions. Briefly, cDNA was generated using the oligo-dT primer to enrich for mRNA,
350 followed by the tagmentation of the cDNA (Illumina Nextera XT) to generate Illumina-compatible
351 RNA-seq libraries. The libraries were amplified by 12 PCR cycles and size-selected for an average
352 insert size of 150 bp and sequenced as 100 bp paired-end reads using Illumina Nova-Seq. Sequence
353 reads were mapped to the reference mouse genome (mm10) using STAR 2.5.1b [54] with
354 parameter --quantMode GeneCounts to generate read counts. The edgeR (Bioconductor package)
355 [55] was used to identify differentially expressed (DE) genes using gene models for mm10
356 downloaded from UCSC (www.genome.ucsc.edu). The abundance of gene expression was
357 calculated as count-per-million (CPM) reads. Genes with less than one CPM in at least three
358 samples were filtered out. The filtered libraries were normalized using the trimmed mean of M-
values (TMM) normalization method [56]. P-values were corrected for multiple testing using the
False Discovery Rate (FDR) approach. Gene set enrichment analyses (GSEA) were performed
using the fgsea R package [57]. Genes were ranked based on the fold-change and the datasets were
downloaded from the GSEA website (https://www.gsea-msigdb.org/gsea/).

363

364 Immunoprecipitation

365 Mouse embryonic stem cell line (mESC) was cultured on gelatin-coated plates and maintained in
366 Dulbecco's Modified Eagle Medium (DMEM) complemented with 10% heat-inactivated fetal
367 bovine serum, penicillin (0.2 U/mL), streptomycin (0.2 µg/mL) and L-glutamine (0.2 µg/mL)
368 (Gibco, Waltham, Massachusetts, United States) and supplemented with recombinant mouse
369 Leukemia Inhibitory Factor (LIF, 20 U/ml, Millipore). Cultured mESCs at 60% confluency were
370 washed with PBS twice before the preparation of total lysate. Total protein lysates were prepared
371 using Pierce IP lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitors
372 (Complete Ultra Tablets, Roche) and Pierce Universal Nuclease (Thermo Fisher Scientific). Lysate
373 was cleared by centrifugation at 20 x g for 10 min at 4°C, and incubated rotating end-over-end at
374 4°C with anti-IgG, anti-ZC3H11A or anti-THOC2 antibodies in Protein LoBind 2-ml tubes
375 (Eppendorf). Thereafter, 30 µg of Dynabeads Protein G (Thermo Fisher Scientific) was added to
376 each tube and incubated for 30 min at room temperature, followed by washing three times with
377 Pierce IP lysis buffer. The co-IP proteins were eluted from the magnetic beads by adding 50 µg of
378 elution buffer (5% SDS, 50mM TEAB, pH 7.55) and heat denaturation for 5 min at 90 °C. The
379 eluted co-IP proteins were used for western blot and Mass spectrometric analysis. co-IP
380 experiments were performed in four replicates.

381
382 **Immunoblot analysis**

383 Equal volumes (5 µg) of the prepared co-IPs were separated by SDS-PAGE (4–15%, Bio-Rad)
384 and transferred to PVDF membranes (Millipore). StartingBlock buffer (Thermo Fisher Scientific)
385 was used to block the membrane before the primary anti-ZC3H11A, anti-THOC2 or anti-ALYREF
386 antibodies (1:1000) were added. Proteins were visualized and detected by the Odyssey system (LI-
387 COR).

388

389 **Protein clean-up and digestion**

390 The co-IPs were cleaned up and prepared for mass spectrometry quantification using the S-Trap
391 column method [58]. First, the eluted co-IPs were treated by TCEP (5 mM) to reduce disulfide
392 bonds, followed by adding methyl methanethiosulfonate (MMTS) to a final concentration of 15
393 mM to alkylate cysteines. Thereafter, the lysate was acidified by adding phosphoric acid to a final
394 concentration of 1.2%. The acidified lysate was added to an S-Trap microcolumn (Protifi,
395 Huntington, NY) containing 300 µl of S-Trap buffer (90% MeOH, 100 mM TEAB, pH 7.5) and
396 centrifuged at 4000 × g for 2 min. The S-Trap microcolumn was washed twice with S-Trap buffer.
397 The columns were transferred to new tubes and incubated with 10 ng/µL sequencing-grade trypsin
398 (Promega) overnight at 37°C. The digested proteins were eluted by centrifugation at 4000 × g for
399 1 min with 50 mM TEAB, 0.2% formic acid (FA), followed by 50% acetonitrile (ACN)/0.2% FA,
400 and finally 80% ACN/0.1% FA. The eluted peptides were dried down in a vacuum centrifuge
401 (ThermoSavant SPD SpeedVac, Thermo Fisher Scientific), and finally dissolved in 1% FA.
402 Digested peptides were thereafter desalted by StageTips (Thermo Fisher Scientific) according to
403 the manufacturer's instructions, and subsequently dissolved in 0.1% FA.

Liquid chromatography and mass spectrometry
The dissolved peptides were quantified by mass spectrometry as previously described [17].

Briefly, a Thermo Scientific EASY-nLC 1000 liquid chromatography system coupled with an Acclaim PepMap 100 (2 cm x 75 µm, 3 µm particles, Thermo Fisher Scientific) pre-column in line with an EASY-Spray PepMap RSLC C18 reversed phase column (50 cm x 75 µm, 2 µm particles, Thermo Fisher Scientific) was utilized to fractionate the peptide samples. The eluted peptides were analyzed on a Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer, operated at a Top Speed data-dependent acquisition scan mode, ion-transfer tube temperature of 275°C, and a spray voltage of 2.0 kV.

413

## 414 Mass spectrometric data analysis

Data analysis of raw files was performed using MaxQuant software (version 1.6.4) and the Andromeda search engine [59, 60], with the following parameters: cysteine methyl methanethiosulfonate (MMTS) as a static modification and methionine oxidation and protein N-terminal acetylation as variable modifications. First search peptide MS1 Orbitrap tolerance was set to 20 ppm, and iontrap MS/MS tolerance was set to 0.5 Da. Peak lists were searched against the UniProtKB/Swiss-Prot *Mus musculus* proteome database (UP000000589, version 2019-04-01) with a maximum of two trypsin miscleavages per peptide. The MaxQuant contaminants database was also utilized. A decoy search was made against the reversed database, with the peptide and protein false discovery rates both set to 1%. Only proteins identified with at least two peptides of at least 7 amino acids in length were considered reliable. The peptide output from MaxQuant was filtered by removing reverse database hits, potential contaminants, and proteins only identified by site (PTMs).
Intensity values were used to determine the protein abundance. First, proteins with missing values in more than one replicate in at least one group were filtered out. Thereafter, the filtered intensities were normalized using the variance stabilizing normalization (vsn) method [61] and followed by the imputation of missing values with the deterministic minimal value approach (MinDet) [62] to replace the missing values in the normalized intensities. The normalized intensities were fitted to a linear model and the empirical Bayes moderated t-statistics and their associated *P*-values were used to calculate the significance of differential enriched proteins [63, 64]. The *P*-values were adjusted for multiple testing using the Benjamini-Hochberg procedure [65]. Proteomics data was visualized using the ggplot R-package and Cytoscape v3.8.2.

### Crosslinking immunoprecipitation sequencing (CLIP-seq)

Cultured mESCs were cross-linked using a 254 nM UV crosslinker with an energy setting of 400 mJ/cm². The cross-linked cells were collected in ice-cold PBS with a cell scraper and aliquoted in 1.5 ml tubes (25 million cells/ml). The CLIP-seq library preparation was performed as indicated [66]. Briefly, the total lysate was digested with RNase-I and immunoprecipitated with 10 µg of the following antibodies: anti-ZC3H11A (HPA028526 and HPA028490, Atlas Antibodies), anti-ALYREF (ab202894, Abcam) or anti-IgG (ab37415, Abcam). The IP-RNA complexes were loaded on a 4–12% Bis-Tris (Bio-Rad) gel, transferred to a nitrocellulose membrane, and the bands above 75 kDa for each lane were cut. Extracted RNA molecules from the membrane were used for Illumina library construction as indicated [66]. CLIP-seq reads were trimmed out using *trim_galore* with the criteria to remove reads with low quality and shorter than 15 bp. The trimmed reads were aligned to the mouse reference genome mm10 using STAR aligner with end-to-end options --alignEndsType EndToEnd. The CLAM workflow were used for peak calling and
counting the fold enrichment of IP vs IgG control [67]. The identified peaks with adjusted *P*-value <0.01 were annotated to the mouse mm10 genome using the *peak_annotator* function from CLAM. HOMER software was used for motif finding using the findMotifsGenome.pl script with default parameters for RNA motifs [68].

## Quantitative RT-PCR

Total RNA was extracted using the RNeasy Mini kit (Qiagen) and the samples were treated with DNase I to eliminate genomic DNA. The High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used to generate cDNA from RNA. Quantitative PCR analysis was performed in ABI MicroAmp Optical 384-well Reaction plates on an ABI 7900 real-time PCR instrument using SYBR gene expression reagents (Applied Biosystems). The amplification and detection of each gene was performed using forward and reverse primers for *Zc3h11a*, F: TGCCTAATCAGGGAGAAGACTG, R: AGCTTCACAGTGACGGAATG and *Actb* as a housekeeping gene F: CTAAGGCCAACCGTGAAAAG, R: ATCACAATGCCTGTGGTACG.

## Derivation of ESCs

E3.5 blastocysts were collected from heterozygous matings. They were plated individually on a layer of Mitomycin C inactivated mouse embryonic fibroblasts (feeder layer) in 4-well plates, in naïve ESC medium. This medium was composed of Chemically Defined Medium (CDM) supplemented with LIF (700 U/ml), PD0332552 (1 µM) and CHIR99201 (final 3 µM) (2i/Lif CDM) [69]. After 4-5 days, the blastocysts have attached and outgrowths formed. Individual outgrowths were dislodged from the feeders, dissociated in Tryple Select (Invitrogen) and the single cell suspension was plated in 4-well plates on fresh feeders. ESC colonies appeared within
the following days and were individually picked, dissociated and replated on feeders in 2i/Lif
473

CDM. The procedure was repeated a few times, until stable expansion of the ESCs that allows
474

passaging using trypsin and removal of feeders, replaced by plate coating with gelatin 0.2% and
475

serum.
476

477

478 Immunostaining of ZC3H11A on pre-implantation embryos

Mouse CD1 embryos were collected at 4-cell (E1.5) and blastocyst (E3.5) stage in M2 medium
479
(Sigma) by oviduct and uterine flushing, respectively. They were fixed in 2% PFA for 20 min,
480
followed by permeabilization by 1% Triton-X100, for 30 min. Permeabilized samples were
481
blocked with 1% BSA in PBS for 40 min, followed by incubation with primary antibodies in 1%
482
BSA overnight at 4°C. The day after, samples were washed by PBS and incubated with the
483
fluorophore conjugated secondary antibodies (Jackson ImmunoResearch) for an hour at room
484
temperature. Samples were then washed and stained by DAPI for nuclei staining. Samples were
485
mounted in Vectashield mounting agent (Vectorlabs, H1000). Embryos were imaged by a Zeiss
486
LSM710 confocal microscope. Antibodies used were anti-SC35 (ab11826, Abcam, 1:250) and
487
anti-ZC3H11A (HPA028526, Atlas Antibodies, 1:300)
488

489

490 Author contributions

SY and LA conceived the study. SY performed experimental and bioinformatic analysis. AJ, VB
491
and JO performed experiments on embryos and ESC derivation with contribution from SY. SY
492
and ML performed mass spectrometry analysis. SY, AJ and LA wrote the paper with input from
493
ML. All authors approved the final version before submission.
494

495
496 Acknowledgments

497 This project was funded by the Swedish Research Council (2017-02907) and the Knut and Alice
498 Wallenberg Foundation (KAW 2017.0071), as well as by the French REVIVE Labex
499 (Investissement d'Avenir, ANR-10-LABX-73). Sequencing was performed by the SNP&SEQ
500 Technology Platform in Uppsala. The Swedish facility is part of the National Genomics
501 Infrastructure (NGI) Sweden and Science for Life Laboratory. The SNP&SEQ Platform is also
502 supported by the Swedish Research Council and the Knut and Alice Wallenberg Foundation.
503 Animal experiments performed in France were done in INRAE Infectiology of Fishes and Rodents
504 Facility (IERP-UE907, Jouy-en-Josas Research Center), which belongs to the National Distributed
505 Research Infrastructure for the Control of Animal and Zoonotic Emerging Infectious Diseases
506 through In Vivo Investigation (EMERG'IN, doi: 10.15454/1.5572352821559333E12). Fluorescent
507 images were acquired in the French ISC MIMA2 (Microscopy and Imaging Facility for Microbes,
508 Animals and Foods, doi: 10.15454/1.5572348210007727E12).
509

509

510 Additional information

511 Competing interests

512 The authors declare no competing interests.

513 Ethics statement

514 Animal procedures were carried out according to the rules and regulations of the Swedish Animal Welfare
515 Agency and French national rules on Ethics and Animal Welfare in the Animal Facility; and were in
516 compliance with the European Communities Council Directive of 22 September 2010 (2010/63/EU).
517 This work was approved by the French Ministry of Higher Education, Research, and Innovation (n°15-55
518 &21-01) and the local Ethical Committee (INRAE Jouy-en-Josas Centre). The study was carried out in
519 compliance with the ARRIVE guidelines.
520

521 **Data availability**

522 The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the

523 PRIDE partner repository with the accession numbers (to be added). The RNA-seq reads have been

524 submitted to the sequence read archive (http://www.ncbi.nlm.nih.gov/sra) with the accession numbers (to

525 be added).

526
527 **Figure legends**

528 **Figure 1. Development of Zc3h11a<sup>-/-</sup> mouse models.** (A) The Zc3h11a locus showing the 529 targeted exons for generating Zc3h11a<sup>-/-</sup> mouse models. (B) Two CRISPR/cas9 guide RNAs were 530 used to delete exon 3 of Zc3h11a. Scissors indicate the location of the gRNAs and the length of 531 deleted sequences. (C) Two homology arms were used to insert loxP sites flanking exon 2. The 532 conditional knock-out mice were crossed with mice expressing Cre in germ line to eliminate the 533 sequences between the loxP sites resulting in the elimination of the entire exon 2 coding sequences. 534 (D and E) Genotyping of the offspring of Zc3h11a heterozygous matings (Zc3<sup>+/-</sup> X Zc3<sup>+/-</sup>) using 535 the CRISPR/cas9-based KO mouse model (D) and the loxP/Cre-based KO mouse model (E). The 536 total numbers of genotyped mice at week 4 are indicated.

537
538 **Figure 2. Ablation of Zc3h11a leads to early embryo degeneration.** (A, top) Schematic 539 illustration showing embryo stages and time points of collecting embryos for genotyping of 540 Zc3h11a. (A, low) PCR genotyping of collected embryos at the above time points. (B) Morphology 541 of collected embryos at E6.5 from Zc3h11a heterozygous mating (Zc3<sup>+/-</sup> X Zc3<sup>+/-</sup>).

542
543 **Figure 3. Cellular localization of ZC3H11A in early embryonic cells.** (A) Immunofluorescence 544 staining of mouse embryos using anti-ZC3H11A and anti-SRSF2 (SC35) antibodies at 2-cell (top) 545 and blastocyst stage (middle and bottom). Middle panel shows a z-projection of the whole 546 blastocyst while the bottom panel is a mid-section through the ICM. (Scale bar: 20 µm). (B) 547 Fluorescent intensity profile of the ZC3H11A signal and paraspeckle marker SRSF2 signal across 548 paraspeckles in an ICM nucleus showing the co-localization of ZC3H11 to paraspeckles. (Scale 549 bar: 5 µm.). (C) Expression of Zc3h11a and Srsf2 measured by smartseq2 single cell RNA-seq. 550 Re-analysis of data from Deng *et. al.* [19].
Figure 4. Transcriptome analysis reveals dysregulated pathways in Zc3h11a<sup>-/-</sup> embryos. (A, left) Dissected inner cell mass (ICM) cells were used for RNA-sequencing. (A, right) Principle component analysis (PCA) of RNAseq data from embryonic parts at E4.5. Dots represent individual embryos and colors represent different genotypes. (B-D) Gene set enrichment analysis (GSEA) of ranked DE genes in Zc3h11a<sup>-/-</sup> embryos using hallmark gene sets. (B-C, below) Heatmaps showing the expression of the genes contributing to the above pathways and found significantly down-regulated in Zc3h11a<sup>-/-</sup> embryos (FDR <0.05). (E) Heatmap showing the expression of the genes contributing to fatty acid metabolism pathway and significantly down-regulated in Zc3h11a<sup>-/-</sup> embryos (FDR <0.05). FDR: false discovery rate, NES: normalized enrichment score.

Figure 5. Down-regulated genes in Zc3h11a<sup>-/-</sup> embryos are ICM-related. (A-B) GSEA of ranked DE genes in Zc3h11a<sup>-/-</sup> embryos with positive enrichment for the P53 pathway (A) and autophagy-related genes (B) among the up-regulated genes in Zc3h11a<sup>-/-</sup> embryos (FDR <0.05). (C) Heatmap of down-regulated genes in Zc3h11a<sup>-/-</sup> embryos (FDR <0.05) and their expression profile during embryonic stages as indicated. Re-analyzed data from GSE76505 and E-MTAB-2950. (D) Expression level of the indicated genes as count per millions (CPM). *, ** and *** correspond to FDR< 0.05, 0.01 and 0.001, respectively.

Figure 6. ZC3H11A binds RNA-export TREX complex proteins in mESC. (A) Schematic illustration of co-immunoprecipitation (co-IP) mass-spectrometry experiments using anti-ZC3H11A, anti-THOC2 and anti-IgG antibodies and mouse embryonic stem cells (mESCs). (B) Heatmap of the interacting partners to ZC3H11A (adjusted P <0.05). Data presented as log
intensities of four replicates. Proteins associated with the TREX complex and mRNA export are in bold. (C) Volcano plot showing the enrichment of co-IP proteins from anti-ZC3H11A/anti-IgG. (D) Western blot of reciprocal co-IP using anti-ZC3H11A, anti-THOC2 and anti-IgG antibodies and probed with the indicated antibodies. Asterisk indicates a cut in the western blot membrane. (E) Log intensities of the ZC3H11A and THOC proteins. (F) Log intensities of FYTTD1 (UAP56) and the polyadenylation factor PABPN1. (G) Log intensities of proteins interacting with ZC3H11A independent of THOC2 and the TREX complex. *, **,*** and **** correspond to adjusted *P* <0.05, 0.01, 0.001 and 0.0001, respectively. ns: not significant.

**Figure 7. CLIP-seq analysis of ZC3H11A RNA targets in mESCs.** (A) Distribution of the proportion of ZC3H11A CLIP-seq mapped reads over the various elements of a gene in mESC using two anti-ZC3H11A antibodies and an anti-IgG control antibody. (B, top) Predicted motifs for ZC3H11A binding. (B, bottom) The overlap between differential down-regulated genes in KO embryos and predicted ZC3H11A CLIP-seq targets. (C) Gene ontology analysis of the down-regulated genes with ZC3H11A binding sites. (D) Heatmap of the down-regulated genes with ZC3H11A binding sites. (E) The visualization of CLIP-seq reads and their distribution over the indicated genes. Black arrows indicate the direction of transcription from 5' UTR to 3' UTR.

**Figure 8. Phenotype characterization of conditional Zc3h11a-KO mice.** (A) The loxP-Zc3h11a mouse model was crossed with mice containing a fusion of a mutated estrogen receptor and Cre recombinase (Cre-ER). The mice were bred to obtain two genotypes of homozygous loxP-Zc3h11a mice (Zc3<sup>loxP/loxP</sup>), one with one copy of Cre-ER (CRE.ER<sup>+</sup> Zc3<sup>loxP/loxP</sup>) and the other with null Cre-ER (CRE.ER<sup>-</sup> Zc3<sup>loxP/loxP</sup>). These mice were crossed and the offspring were injected with tamoxifen at week 3-4 after birth. The time line indicates the time points of injection and sample
collection for genotyping and phenotyping. (B) Genotyping of the Cre-ER Zc3loxP mice. (C) qPCR analysis of Zc3h11a mRNA expression in bone marrow, liver and spleen tissues from WT and induced Zc3-KO (iKO) mice both injected with tamoxifen. ** and *** correspond to *-test P <0.01 and 0.001, respectively. (D) Western blot analysis of spleen tissues dissected from WT and iKO adult mice. (E) Histology (H&E staining) of small intestine from WT and induced iKO adult mice. (F) Body weight in grams (g), weight of dissected kidney and spleen in milligrams (mg) from WT and induced iKO adult mice. Results are means ± SEM.

## Supplementary information

**Figure S1.** Heatmaps of the normalized expression of genes involved in the P53 pathway (A) and in positive regulation of autophagy (B).

**Figure S2.** (A) Heatmap of up-regulated genes (A, left) and down-regulated genes (A, right) in Zc3h11a⁻/⁻ embryos (FDR <0.05) and their expression profile during embryonic stages as indicated. Re-analyzed data from GSE76505 and E-MTAB-2950. (B) Violin plot of the relative expression of inner cell mass (ICM)-related genes and trophectoderm (TE)-related genes (ICM/TE) as detected among the down-regulated and up-regulated genes in Zc3h11a⁻/⁻ embryos (FDR <0.05).

**Figure S3.** (A, top) Chord diagram illustrating the overlap between the interacting partners detected with ZC3H11A and THOC2 co-IPs. (A, bottom) GO analysis of the identified interacting
partners with ZC3H11A and THOC2 co-IPs in mESCs. (B) Network analysis of the identified interacting partners with ZC3H11A and THOC2 based on co-IPs in mESCs. Blue lines represent the interaction detected by our proteomics analysis, and gray lines represent the predicted interaction from the STRING database.

**Figure S4.** (A) Genome-wide distribution of the ZC3H11A CLIP-seq peaks from mESCs across different types of transcripts. (B) Visualization of CLIP-seq reads and their distribution over the *Neat1* gene.

**Figure S5.** Histology staining of intestine, colon and stomach tissues from three WT and three induced ZC3-KO adult mice.

**Table S1.** Differentially expressed genes in *Zc3h11a* knock-out (KO) E4.5 embryos.

**Table S2.** Sequences of PCR primers used for mouse genotyping.

**References**

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2. E. G. Folco, C.-S. Lee, K. Dufu, T. Yamazaki, R. Reed, The proteins PDIP3 and ZC11A associate with the human TREX complex in an ATP-dependent manner and function in mRNA export. *PLoS One* **7**, e43804 (2012).

3. L. Yang, et al., Porcine ZC3H11A Is Essential for the Proliferation of Pseudorabies Virus and Porcine Circovirus 2. *ACS Infect. Dis.* (2022) https://doi.org/10.1021/ACSINFECDIS.2C00150.
647 4. C. G. Heath, N. Viphakone, S. A. Wilson, The role of TREX in gene expression and disease. *Biochem. J.* **473**, 2911–2935 (2016).

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649 5. B. Chi, et al., Aly and THO are required for assembly of the human TREX complex and association of TREX components with the spliced mRNA. *Nucleic Acids Res.* **41**, 1294–1306 (2013).

651 6. K. Dufu, et al., ATP is required for interactions between UAP56 and two conserved mRNA export proteins, Aly and CIP29, to assemble the TREX complex. *Genes Dev.* **24**, 2043–53 (2010).

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<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>Figure 1</figcaption></figure>

<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>B</figcaption></figure>

HALLMARK_GLYCOLYSIS

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>D</figcaption></figure>

HALLMARK_FATTY_ACID_METABOLISM

<figure><img src="image_16.png" /><figcaption>C</figcaption></figure>

HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /><figcaption>Figure 5</figcaption></figure>

<figure><img src="image_23.png" /></figure>
<figure><img src="image_24.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>Figure 6</figcaption></figure>
A

<figure><img src="image_31.png" /></figure>

B

<figure><img src="image_32.png" /></figure>

C

<figure><img src="image_33.png" /></figure>

D

Down-reg genes with ZC3H11A binding

<figure><img src="image_34.png" /></figure>

E

CLIP-seq of mESC

<figure><img src="image_35.png" /><figcaption>Figure 7</figcaption></figure>
<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /><figcaption>Figure 8</figcaption></figure>
A
HALLMARK_P53_PATHWAY

<figure><img src="image_44.png" /></figure>

B
POSITIVE_REGULATION_OF_AUTOPHAGY

<figure><img src="image_45.png" /><figcaption>Figure S1</figcaption></figure>
A

Expression of up-reg genes in ZC3-/- embryos

<figure><img src="image_46.png" /></figure>

Expression of down-reg genes in ZC3-/- embryos

<figure><img src="image_47.png" /></figure>

B

<figure><img src="image_48.png" /><figcaption>Figure S2</figcaption></figure>
<figure><img src="image_49.png" /><figcaption>Figure S3</figcaption></figure>
<figure><img src="image_50.png" /><figcaption>A
ZC3H11A target transcripts</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>Figure S4</figcaption></figure>
<figure><img src="image_52.png" /><figcaption>Figure S5</figcaption></figure>