Small nucleolar RNA Snora61 drives self-renewal of intestinal stem cells via initiation of Lgr5 transcription

Domain: neuroscience
Published: 2026-04-07
Editorial outcome: major_revision

Abstract
Intestinal epithelium relies on intestinal stem cells (ISCs) for rapid and precise tissue
replenishment to maintain gut normal function. The self-renewal maintenance of ISCs is finely
regulated by multiple stemness factors and signaling pathways. However, the transcription
mechanisms of some key stemness factors remain poorly understood. Here we identify that small
nucleolar RNA Snora61 is highly expressed in ISCs. Snora61 is mainly distributed in the nucleoplasm.
Snora61 knockout impairs ISC self-renewal and intestinal regeneration. Mechanistically, Snora61
binds to the promoter region of Lgr5 gene and engages with RNA-binding protein RBMX to recruit
HMGB2 onto Lgr5 promoter, leading to Lgr5 transcription and expression. Snora61 promotes the self-
renewal of small intestinal stem cells, which in turn enhances the proliferation of differentiated epithelial
cells, thereby contributing to the maintenance of intestinal homeostasis. Conversely, Snora61
knockout causes reduced LGR5 expression. Deletion of Lgr5 with Snora61 displays more severely
impaired ISC self-renewal and intestinal regeneration. Our findings reveal a regulatory mechanism of
Lgr5 transcription underlying ISC self-renewal maintenance.

Introduction
The intestinal epithelium, as a digestive tissue that directly contacts external environment, is
subject to continuous mechanical, chemical, and biological damage while absorbing metabolic
substances1. Once the epithelium is breached, it may lead to wear and tear of the underlying tissue,
even systemic infection2. Therefore, continuous tissue replacement is crucial for maintaining intestinal
homeostasis. The mouse intestinal epithelium renews every 3-5 days3. This continuous tissue
replacement is provided by the intestinal stem cells (ISCs) located at the base of the crypts. In the
crypts, the constantly dividing ISCs proliferate and ultimately differentiate into mature intestinal
epithelial cells4. Control of ISC stemness is critical for intestinal homeostasis.

Earlier studies have identified LGR5 as a highly specific marker for ISCs5. Extensive reports have
demonstrated that the self-renewal of ISCs is regulated by various signaling molecules and pathways,
such as Wnt, Notch, Hippo, EGF, bone morphogenetic protein, interleukin, and metabolic signals6. In
the Wnt signaling pathway, LGR5 binds R-spondin to maintain abundance of Frizzled receptors on cell
membrane by sequestering the E3 ubiquitin ligases RNF43 and ZNRF3, thereby activating Wnt
signaling7. LGR5, acting as a critical receptor for ISCs, has been well characterized in terms of its
function. However, the precise mechanisms driving expression of LGR5 in ISCs remains elusive.

Small nucleolar RNAs (snoRNAs) are short non-coding RNAs8. SnoRNAs are typically classified
into two types, C/D box and H/ACA box snoRNAs, which regulate ribosome biogenesis by guiding 2’-
O-methylation and pseudouridylation respectively9. Recent studies have increasingly revealed that
some snoRNAs are also involved in a broader range of transcriptional and post-transcriptional
regulatory processes, including modulating mRNA levels10, participating alternative splicing11,
acetylating rRNA12, competing ribosomal subunit binding sites13, and regulating protein secretion and
metabolic activities14. However, how snoRNAs regulate LGR5 in ISC maintenance is unknown.

HMGB2 belongs to the HMG-box protein family, which is a subgroup of high-mobility group (HMG)
superfamily15. HMGB2 participates in numerous cellular functions such as transcription16 and
chromatin remodeling17. It has been reported that HMGB2 exerts regulatory functions in various types
of stem cells. For example, HMGB2 is intricately linked with the activation of human neural stem cells
from a dormant state to an active proliferative phase18. The interaction between HMGB2 and OCT4
contributes to pluripotency gene expression in mouse embryonic stem cells19. In addition, HMGB2
regulates proliferation and differentiation of human embryonic stem cells (hESCs) and progenitor cells
by modulating telomerase activity20. Moreover, HMGB2 participates in self-renewal maintenance of
hematopoietic stem cells through regulation of latexin transcription21.

Here we identify Snora61 as an important noncoding RNA in the regulation of ISC self-renewal
maintenance. Snora61 associates with RBMX to recruit HMGB2 onto Lgr5 promoter, leading to Lgr5
transcription and expression.

Results
Snora61 is highly expressed in ISCs
To investigate expression profiles of snoRNAs in ISCs, we utilized Lgr5-eGFP mice to isolate
LGR5+ ISCs and LGR5− intestinal epithelial cells (IECs) via flow cytometry, followed by snoRNA
microarray assay. Through analysis and screening, we identified that H/ACA snoRNA Snora61 was
highly expressed in ISCs (Fig. 1a, b). In addition, we designed shRNAs against the top ten enriched
snoRNAs and found that knockdown of Snora61 most severely impaired organoid formation capacity
(Fig. 1c and Supplementary Fig. 1a, b). Snora61 is located within Snhg12 gene on mouse
chromosome 4 (Supplementary Fig. 1c). The Snhg family, which encodes snoRNAs, comprises non-
coding genes22. Human SNORA61 shared a similar genomic localization with its mouse counterpart
and exhibited 73.68% sequence homology (Supplementary Fig. 1c, d). Knockdown of SNORA61 in
human intestinal organoids exhibited similar inhibitory effect on organoid formation (Supplementary
Fig. 1e). Actually, the Snhg12 gene encodes four snoRNAs: Snora16a, Snora44, Snora61, and
Snord99. We confirmed that among these four snoRNAs, only Snora61 was enriched in LGR5+ ISCs
(Supplementary Fig. 1f). To further determine tissue-specific expression of Snora61, we performed
fluorescence in situ hybridization (FISH) staining of frozen sections from fetal mice. We observed that
Snora61 was highly expressed in small intestines and spleen (Fig. 1d). Northern blot and RT-qPCR
showed similar observations (Fig. 1e and Supplementary Fig. 1g). In addition, Snora61 was
specifically enriched at the tips of budding structures in intestinal organoids, corresponding to the crypt
regions of small intestines23 (Fig. 1f). This observation was consistent with our staining results from
intestinal sections. Snora61 was predominantly localized in the base of crypts and co-localized with
LGR5+ cells (Fig. 1g, h).
To investigate subcellular localization of Snora61, we performed subcellular fractionation and
FISH staining. We found that Snora61 was predominantly distributed in the nucleoplasm (Fig. 1i-k and
Supplementary Fig. 1h). Collectively, Snora61 is highly expressed in ISCs and mainly distributed in
the nucleoplasm.

Snora61 knockout impairs self-renewal of ISCs
To explore the role of Snora61 in ISC self-renewal maintenance, we generated Snora61
knockout (Snora61−/−) mice through CRISPR/Cas9 technology. Snora61 deletion was confirmed by
genotyping and DNA sequencing (Supplementary Fig. 2a, b). Deletion of Snora61 did not influence
expression levels of Snora16a, Snora44, or SNnord99 (Supplementary Fig. 2c). We found that
Snora61−/− mice exhibited shorted intestine length (Fig. 2a), while Snora61−/− mice displayed normal
sizes and morphology of other organs, including brain, heart, liver, spleen, kidneys, and testis
(Supplementary Fig. 2d). As expected, Snora61 knockout caused impaired organoid formation, while
Snora61 overexpression rescued organoid growth and budding (Fig. 2b and Supplementary Fig. 2e).
However, knockdown of Snora16a, Snora44, or Snord99 did not affect organoid formation
(Supplementary Fig. 2f). Moreover, Snora61−/− mice demonstrated reduced crypt numbers across all
regions of small intestines (duodenum, jejunum, and ileum) (Fig. 2c). In addition, small intestines of
Snora61−/− mice showed decreased cell proliferation (Fig. 2d). Consistently, intestinal organoids
derived from Snora61−/− mice also displayed markedly reduced cell proliferation during organoid
formation (Fig. 2e and Supplementary Fig. 2g). We next treated Snora61+/+ and Snora61−/− mice with
8 Gy X-ray irradiation for radiation-induced damage. We noticed that Snora61−/− mice displayed
remarkable inhibition of intestinal regeneration by H&E staining compared with that of littermate
Snora61+/+ mice (Fig. 2f). In parallel, Snora61−/− mice exhibited more severe body weight loss and a
higher disease activity index (DAI) score (Supplementary Fig. 2h, i and Supplementary Table 5).
We also examined the relevant phenotypes in heterozygous mice. Expression levels of Snora61
in Snora61+/− mice were approximately half of those in Snora61+/+ mice (Supplementary Fig. 2j). We
observed analogous but less severe stemness suppression in Snora61+/− mice compared to
Snora61−/− mice, including shortened small intestine and villus length, reduced crypt number, and
impaired organoid formation ability (Supplementary Fig. 2k-m). Taken together, Snora61 is required
for self-renewal maintenance of ISCs.

Snora61 initiates Lgr5 transcription in ISCs
Snora61 canonically functions to mediate pseudouridylation at position 2263 of 28S rRNA
(Supplementary Fig. 3a). To determine whether Snora61 mediated pseudouridylation regulated ISC
stemness, we performed N-cyclohexyl-N’-(2-morpholinoethyl) carbodiimide methyl-p-toluene
sulfonate (CMC) treatment to assess levels of 28S rRNA U2263 pseudouridylation across different
experimental groups (Supplementary Fig. 3b). We found that Snora61 deletion did not affect
abundance of 28S rRNA (Supplementary Fig. 3c). However, Snora61 deletion caused markedly
reduced levels of U2263 pseudouridylation. whereas overexpressing wild-type (WT) Snora61 could
rescue U2263 pseudouridylation but not mutant Snora61 (Supplementary Fig. 3d, e). Of note,
overexpressed WT or mutant Snora61 in Snora61−/− ISCs could restore organoid growth
(Supplementary Fig. 3f), suggesting that U2263 pseudouridylation was not involved in the
maintenance of ISC self-renewal. We concluded that Snora61-mediated ISC self-renewal is
independent of its canonical pseudouridylation function.
We next carried out RNA-seq from ISCs of Snora61+/+ and Snora61−/− mice. GO analysis revealed
that the downregulated genes were significantly enriched in pathways related to intestinal
development, cell proliferation/differentiation, and gene transcription (Fig. 3a). We hypothesized
that Snora61 knockout may impair transcription of key stemness-related genes. Consequently, we
analyzed differential expression levels of main stemness-related genes and found that Lgr5 was most
downregulated gene in Snora61−/− ISCs (Fig. 3b and Supplementary Fig. 3g). Lgr5 and its downstream
target genes showed various degrees of downregulation, which was particularly evident during post-
irradiation repair (Fig. 3c, d). Consequently, the gene expression profile in Snora61−/− ISCs showed
significantly reduced enrichment in the Wnt signaling pathway post irradiation (Fig. 3e). In addition,
ChIRP-seq analysis showed that Snora61 was enriched on the Lgr5 promoter region (Fig. 3f).
Sequence alignment indicated complementary base-pairing between Snora61 and the enriched
genomic segment (Fig. 3g). This binding and enrichment were further confirmed by ChIRP-qPCR and
dot-blot assays, while mutation of the binding sites of Snora61 abolished the engagement (Fig. 3h, i).
These results indicate that Snora61 binds to the Lgr5 promoter via complementary base-pairing
sequences.
Dual luciferase reporter assays demonstrated that Snora61 promotes Lgr5 transcription, and this
effect is dependent on the -2000 ~ -1800 bp binding region (Fig. 3j). Consequently, Snora61 deletion
suppressed chromatin accessibility and transcription activity of Lgr5 gene (Fig. 3k, l). In parallel,
Snora61 deletion dramatically decreased H3K4me3 levels of the -2000 ~ -1800 bp region of Lgr5
promoter, whereas increased H3K27me3 levels (Fig. 3m, n). The impaired expression in Snora61−/−
ISCs was further validated by immunoblotting (Fig. 3o). In addition, we crossed Snora61+/+ and
Snora61−/− mice with Lgr5-eGFP mice to evaluate proportions of ISCs. We observed that proportions
of LGR5+ cells were remarkably decreased in Snora61−/− mice (Fig. 3p). Consistently, LGR5
expression was lowly expressed in Snora61−/− mice crypts and organoids (Supplementary Fig. 3h, i).
In addition, LGR5 expression was relatively downregulated in Snora61+/− ISCs and in human intestinal
organoids with SNORA61 knockdown (Supplementary Fig. 2j and Supplementary Fig. 1e).
Furthermore, we monitored dynamic expression of Lgr5 and Snora61 during intestinal repair post
irradiation. Time-course analysis revealed that with initial synchronous decline with Lgr5 due to ISCs
loss, Snora61 expression rebounded ahead of Lgr5 recovery (Supplementary Fig. 3j). By contrast,
mutant Snora61 could not restore organoid formation ability and LGR5 expression in Snora61−/−
organoids (Fig. 3q and Supplementary Fig. 3k, l). Collectively, these results indicate that Snora61
enriches on the Lgr5 promoter to initiate Lgr5 transcription in ISCs.

Snora61 associates with RBMX in ISCs
To further explore the molecular mechanism by which Snora61 promoted Lgr5 transcription, we
performed RNA pull-down assays (Fig. 4a). Through mass spectrometry analysis of the two most
prominent differential bands (located around 42 kDa and 30 kDa), we identified a set of binding protein
candidates for Snora61 (Supplementary Table 6). Based on subcellular localization of these
candidates and their regulatory effects on Lgr5 expression, we ultimately selected RBMX for further
validation. RBMX exhibited clear nuclear localization, and its knockdown suppressed Lgr5 expression
(Supplementary Fig. 4a-c). This interaction was further validated by Western blot and RNA
immunoprecipitation assays (Fig. 4b, c). RBMX, an RNA-binding protein, contains an RNA recognition
motif (RRM) domain. Truncation mapping demonstrated that Snora61 bound to the RRM domain of
RBMX (Fig. 4d). Fluorescence staining revealed that RBMX was co-localized with Snora61 in ISCs
and intestinal organoids (Fig. 4e, f).
To investigate the role of RBMX, we silenced and overexpressed Rbmx in intestinal organoid and
examined the effect of RBMX on organoid growth (Supplementary Fig. 4d). We observed that Rbmx
knockdown inhibited organoid growth in both Snora61+/+ and Snora61−/− ISCs, while Rbmx
overexpression promoted organoid formation (Fig. 4g and Supplementary Fig. 4e). These
observations were verified by Ki67 staining ( Fig. 4h and Supplementary Fig. 4f). As expected, Rbmx
knockdown suppressed LGR5 expression, whereas Rbmx overexpression enhanced LGR5
expression (Fig. 4i and Supplementary Fig. 4g, h). To assess the functional requirement of the RBMX-
Snora61 complex, we performed rescue assays in shRbmx organoids. Expression of an RRM-deletion
mutant of RBMX failed to rescue impaired organoid formation and low Lgr5 expression, unlike its full-
length protein (Fig. 4j and Supplementary Fig. 4i, j). ChIP analysis confirmed RBMX enrichment at the
-2000 ~ -1800 bp region of Lgr5 promoter, which was abrogated by Snora61 knockout (Fig. 4k, l). Of
note, Rbmx knockdown or overexpression did not affect Snora61 abundance (Supplementary Fig. 4k).
Similarly, Snora61 deletion did not influence expression of RBMX (Supplementary Fig. 4l). Taken
together, RBMX associates with Snora61 in ISCs and cooperatively regulates LGR5 expression.

Snora61-RBMX recruits HMGB2 onto Lgr5 promoter to initiate its transcription
To further explore how Snora61-RBMX engagement regulated Lgr5 expression, we used anti-
RBMX antibody to go through ISC lysates and the most prominently differential band (around 24 kDa)
was conducted by mass spectrometry (Fig. 5a and Supplementary Table 7). After evaluating
subcellular localization and transcriptional activity of these candidate proteins, we ultimately identified
HMGB2 as an interacting protein of RBMX (Supplementary Fig. 5a). The interaction between RBMX
and HMGB2 was further validated by co-IP assay (Fig. 5b, c). Of note, the abundance and interaction
of RBMX and HMGB2 in ISCs did not depend on Snora61 (Fig. 5d). Snora61, RBMX, and HMGB2
were co-localized in the nucleus of ISCs and intestinal organoids (Fig. 5e-g). HMGB2, a co-
transcription factor, regulates gene transcription by binding to chromatin and recruiting other
transcription factors onto gene promoters. We depleted Hmgb2 in ISCs (Supplementary Fig. 5b) and
followed by intestinal organoid formation assay. We observed that Hmgb2 silencing inhibited organoid
growth and LGR5 expression in ISCs (Fig. 5h-k and Supplementary Fig. 5c). In addition, we found that
the expression of Snora61 and HMGB2 is mutually independent (Supplementary Fig. 5d, e). Hmgb2
knockdown abrogated Snora61 overexpression increased organoid formation (Supplementary Fig. 5f).
However, overexpression of Hmgb2 failed to restore the impaired organoid formation
and Lgr5 expression in Snora61−/− organoids (Supplementary Fig. 5g, h). These data suggest that
HMGB2 is involved in Snora61-mediated maintenance of ISC stemness.
Through ChIP assay, we observed that HMGB2 was enriched onto the -2000 ~ -1800 bp region
of Lgr5 promoter, while Snora61 deletion abolished this enrichment (Fig. 5l, m). Moreover, through
dual luciferase reporter assay, we noticed that Hmgb2 overexpression promoted Lgr5 transcription
activity, whereas deletion of -2000 ~ -1800 region abrogated this effect (Fig. 5n). We next generated
AAV-mediated Rbmx conditional knockout mice (Supplementary Fig. 6a). In this setting, Hmgb2
overexpression failed to rescue the impaired organoid formation and reduced Lgr5 expression
in Hmgb2 knockdown ISCs (Supplementary Fig. 6b-d). In addition, Rbmx knockout abrogated
enrichment of HMGB2 to the Lgr5 promoter (Supplementary Fig. 6e). Finally, Rbmx knockdown
inhibited Lgr5 transcription activity in dual luciferase reporter assay (Supplementary Fig. 6f and Fig.
5n). Altogether, Snora61 recruits RBMX-HMGB2 complex onto the Lgr5 promoter to initiate its
transcription in ISCs.

LGR5 is required for Snora61-mediated ISC self-renewal and intestinal regeneration
To test the physiological role of LGR5 in maintaining ISC self-renewal, we generated Lgr5
conditional knockout mice using AAV and validated knockout efficiency via Western blot, RT-qPCR
and FACS (Fig. 6a and Supplementary Fig. 7a-c). Lgr5 knockout dramatically reduced the proportions
of Ki67+ cells (Fig. 6b). In addition, Lgr5 knockout also markedly suppressed expression of OLFM4,
another key ISC marker (Supplementary Fig. 7d, e). We noticed that Lgr5 knockout decreased length
of intestinal villi (Fig. 6c). Meanwhile, deletion of Lgr5 with Snora61 exhibited much shorter length of
intestinal villi (Fig. 6c). Consistently, Lgr5 knockout impaired formation and budding of intestinal
organoids (Fig. 6d) and significantly inhibited cell proliferation in intestinal organoids (Supplementary
Fig. 7f). We observed that deletion of Lgr5 with Snora61 showed much more severely abolished
intestinal organoid formation and cell proliferation (Fig. 6d, and Supplementary Fig. 7f). Moreover, Lgr5
knockout obviously impaired intestinal regeneration post irradiation exposure (Fig. 6e). Finally,
deletion of Lgr5 with Snora61 manifested much worse intestinal regeneration post irradiation exposure,
along with more severe weight loss and higher DAI scores (Fig. 6e and Supplementary Fig. 7g, h).
dFz7-21 is an antagonist of Frizzled receptor24. We observed that dFz7-21 treatment abrogated
Snora61 overexpression induced organoid formation capacity in Snora61−/− ISCs (Fig. 6f), suggesting
that LGR5-mediated Wnt signaling participates in Snora61-induced ISC self-renewal maintenance. Of
note, knockdown of SNORA61 in human intestinal organoids with an APC mutation did not affect
organoid growth (Supplementary Fig. 7i). Collectively, these results indicate that LGR5 is required for
Snora61-mediated ISC self-renewal and regeneration.

Methods
Ethics
All experiments in this study were conducted in compliance with relevant ethical regulations and
approved by the Institutional Committee of the Institute of Biophysics, Chinese Academy of Sciences.
Both male and female mice were housed under specific pathogen-free conditions with a 12 h light/dark
cycle, temperature of 25±2°C, and 50% relative humidity. Mice were randomly assigned to
experimental groups, and no sex-dependent differences were observed in any of the analyses. All
animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of
the Institute of Biophysics, Chinese Academy of Sciences. No biases were introduced with respect to
patient sex or gender.

Antibodies and reagents
Anti-Ki67 (Cat# 11-5698-82, Cat# 14-5698-80), 488 Donkey anti-Rabbit, Secondary Antibody
(Cat# A21206), 594 Donkey anti-Rabbit, Secondary Antibody (Cat# A21207) and 488 Donkey anti-
Mouse, Secondary Antibody (Cat# R37114) were purchased from Invitrogen. Anti-EpCAM (CD326)
(Cat# 118211) was purchased from Biolegend. Anti-Olfm4 (Cat# 14369), Anti-H3K4me3 (Cat# 9751T),
anti-H3K27me3 (Cat# 9733), Anti-RBMX (Cat# 14794S) and Anti-Histone H3 (Cat# 4499) were
purchased from Cell Signaling Technology (Danvers, USA). Anti-Nucleolin (Cat# ab129200), Anti-
RBMX (Cat# ab190352) and Anti-HMGB2 (Cat# ab124670) were purchased from Abcam. Anti-
HMGB2 (Cat# 14597-1-AP), Anti-Nucleolin (Cat# 10556-1-AP) and Anti-LGR5 (Cat# 30007-1-AP)
were purchased from Proteintech. Anti-HMGB2 (Cat# K108316P) and Anti-RBMX (Cat# K007062P)
were purchased from Solarbio. Anti-HMGB2 (Cat# WH0003148M5) was purchased from Sigma. Anti-
RBMX (Cat# AF5263) was purchased from Affinity. Anti-EEA1 (Cat# SC-6415) was purchased from
Santa Cruz. Anti-Lamin A/C (Cat# HY-P80204) and Protein A/G Magnetic Beads (Cat# HY-K0202)
were purchased from MCE. Anti-β-actin (Cat# RM2001), Anti-Flag-tag (Cat# RM1002), Goat anti-
mouse IgG (Cat# RM3001) and Goat anti-rabbit IgG (Cat# RM3002) were purchased from Ray
Antibody. All antibodies were used at the manufacturer's recommended concentrations. F-actin
staining kit (Cat# 23122) was purchased from AAT Bioquest. The Dual Luciferase Reporter Gene
Assay Kit (Cat# RG027) and Streptavidin Magnetic Beads (Cat# 23122) were purchased from
Beyotime. The Biotin RNA Labeling Mix (Cat# 11685597910), T7 RNA polymerase (Cat#
10881767001) and Blocking Reagent (Cat# 11096176001) were purchased from Roche. The
Chemiluminescent Nucleic Acid Detection Module (Cat# 89880), NE-PER Nuclear and cytoplasmic
extraction (Cat# 78833) and Supersignal West pico plus (Cat# 34577) were purchased from Thermo.
The Organoid Growth Medium was purchased from Stem cell (Cat# 06005) and BioGenous (Cat#
K2003-HC, Cat# K-260A5). The Matrigel was purchased from Corning (Cat# 356237).

Animals
Villin-Cre, Cas9-KI and Lgr5-eGFP mice were purchased from the Jackson Laboratory. The
Snora61−/− mice were generated by the gene editing facility of Institute of Biophysics using the
CRISPR/Cas9 system. The specific procedure is as follows: the sgRNAs targeting Snora61 were
designed by online CRISPR design tool (https://crispor.gi.ucsc.edu/). The sgRNA sequences are
listed in Supplementary Table 1. Approximately 250 zygotes in the C57BL/6 background were injected
with corresponding single guide RNA (sgRNA) donors and subsequently transferred to the uterus of
pseudo-pregnant ICR females, from which viable founder mice were obtained. After extracting
genomic DNA from the offspring mice, mice with successful knockout were identified by PCR and
next-generation sequencing. Through multiple generations of breeding, stably inherited homozygous
mice were ultimately established. All the mouse strains were C57BL/6 background and maintained
under specific pathogen-free conditions. Male and female mice aged 6 to 10 weeks were used. This
study is compliant with all relevant ethical regulations regarding animal research.

Cell lines
HEK293T and MC38 cells were obtained from ATCC (American Type Culture Collection,
Manassas, VA, USA). HEK293T and MC38 cells were cultured in DMEM medium supplemented with
10% FBS, 100 μg/ml penicillin G and 100 U/ml streptomycin.

Human intestinal tissues and CRC samples
Primary human CRC samples were collected from patients pathologically diagnosed with
colorectal cancer from the Department of General Surgery, Peking University Third Hospital with
informed consent and the approval from the Ethics Committee of Peking University Third Hospital.
The participants were selected randomly, without potential self-selection bias or other biases. Patient
information is presented in Supplementary Table 8. Fresh human tissues/samples were minced into
1 mm3 pieces and then digested with digestion buffer (0.1% IV collagenase, 0.01% DNase, 0.05%
protease dissolved in serum-free DMEM) at 37°C for 30 min. The supernatant was then filtered
through a 100 μm filter and centrifuged at 150 g for 5 min. Organoids were cultured using the obtained
cells with the Human Colonic Organoid Kit (Cat# K2003-HC) / Human Colorectal Cancer Organoid
Kit (Cat# K-2605).

SnoRNAs sequencing and microarray assay
For snoRNAs sequencing, 5 × 105 LGR5+ ISCs and LGR5− IECs were collected from small
intestine crypts from Lgr5-eGFP mice aged 8 weeks by FACS sorting and were detected by confocal
microscopy (purity over 95%). Total RNA was extracted using a standard RNA-extraction protocol
and then snoRNA microarray assay analysis was performed by Yingbio Tech Company.

Small intestine organoid formation assay
Small intestines from indicated mice aged 8 weeks were isolated, cut open longitudinally and
washed using phosphate buffer saline (PBS) several times. The intestinal villus was removed. Then,
intestines were cut into 5 mm pieces, resuspended with Collagenase digestion buffer (1mg/mL
collagenase I in PBS) and incubated for 20 min at 37°C. Tissue pieces were transferred into 10 ml
ice cold PBS in 15 ml tube. Shake vigorously by hand for one minute and collect first elution
components. Repeat for total three times and check crypts under microscope. Usually, crypts were
enriched in the second elution component. Crypts were further filtered through 70 μm cell strainer.
Crypts were suspended with IntestiCult™ Organoid Growth Medium at density of 200 crypts/25 μl,
mixed with Matrigel at 1:1 ratio, seeded into 6-well plate and cultured for 2-3 days. Primary organoids
usually undergo 2-3 serial passages to remove contaminating cells. Organoid imaging or collection
is conducted 48 to 72 hours subsequent to passaging, based on their growth progression.

shRNA knockdown and gene overexpression system
Silencing of indicated genes was performed by short hairpin RNA. shRNAs of indicated genes
were designed on online RNAi designer (https://rnaidesigner.thermofisher.com/rnaiexpress/). 2–3
shRNAs of each target gene were selected and cloned into pSicoR Puro lentivirus vector, and the
shRNAs were listed in Supplementary Table 2 . Genes for overexpression were constructed into pLVX-
IRES-Puro vector. For rescue experiments used in this study, synonymous mutations (i.e., codon
substitutions) were introduced into shRNA-targeted region during design of overexpression plasmids,
which ensured that the original shRNAs did not interfere with rescue effect. Lentiviral vectors were co-
transfected with packaging plasmids psPAX2 and pMD2G into HEK293T cells. Lentiviruses in
supernatants were harvested at 48 and 72 h after transfection and filtered through 0.45 μm filters.
Recombinant lentiviruses were concentrated by ultracentrifugation (25000 rpm, 2 h, 4°C) and
dissolved with Organoid Growth Medium. For gene silencing or overexpression in organoid cells,
organoids from indicated mice intestine were cultured for 3 days. Organoids were broken and collected
by centrifugation. Then broken organoids were resuspended with 50 μl complete organoid media and
1:1 mixed with lentivirus solution containing 6 mg/ml polybrene (Sigma-Aldrich). Mixed with Matrigel
at 1:1 ratio and seeded into 6-well plate, organoids were cultured at 37°C for 3 days. Organoids were
passaged and maintained and gene silencing or overexpression efficiency was analyzed by real-time
qPCR.

CRISPR/Cas9 knockout system
Lgr5/Rbmx-deletion mice were established using CRISPR/Cas9 approaches provided by
Zhang’s lab. The sgRNAs were designed by online CRISPR design tool (https://crispor.gi.ucsc.edu/).
The sgRNA sequences are listed in Supplementary Table 1. In brief, sgRNAs were cloned into pAAV-
U6-gRNA-CBh-mCherry (Addgene, catalog 91947). AAV vectors were co-transfected with packaging
plasmids pHelper and pAnc80 into HEK293T cells. AAV were harvested at 72 h after transfection by
repeated freezing and thawing and filtered through 0.22 μm filters. AAV was injected into Villin-
Cre+Cas9-KI mice aged 6 weeks via the tail vein. The gene knockout efficiency was analyzed by RT-
qPCR and Western blot.

Northern blot
Total RNA was extracted from different organs and tissues from WT mice aged 8 weeks with
standard TRIzol methods, and then subjected to electrophoresis on Urea page gel for 1 h in 0.5×TBE.
Samples were transferred to positively charged NY membranes for 1 h in TBE buffer. After UV cross-
linking (265 nm ultraviolet with energy of 20 0,000 μJ/cm2) and prehybridization, membranes were
incubated with biotin-labeled probes at 65°C for 16–20 h. After washing with washing buffer, biotin
signals were detected with Chemiluminescent Nucleic Acid Detection Module (Cat# 89880) according
to the manufacturer’s instructions. For detecting snoRNAs only, inverse complementary sequences
were used for probes.

Immunofluorescence staining
Organoids or frozen sections were fixed by 4% paraformaldehyde (PFA) for 30 min, and then
permeated by 0.5% triton X-100 for 30 min. After blocking with 10% donkey serum for 1 h, primary
antibodies were added and incubated overnight at 4°C. After washing with PBS, fluorescence-
conjugated secondary antibodies were incubated at RT for 2 h or overnight at 4°C. The DAPI was
incubated at RT for 5 min. After sealing, the samples were observed by confocal microscopy (Nikon
A1R+).

Fluorescence in situ hybridization
Organoids or frozen sections were fixed with 4% PFA for 20 min at room temperature,
permeabilized with PBS containing 0.5% TritonX-100 for 20 min, prehybridized with hybridization
buffer (50% formamide, 5× SSC, 500 ng/μl yeast tRNA, 1× Dehardt’s solution, 500 ng/μl sperm DNA,
50 ng/μl Heparin, 2.5 mM EDTA, 0.1% Tween-20, 0.25% CHAPS) for 1h at 62°C, incubated with
biotinylated or cy5-labeled probes at 62 °C for 2 h, and then washed three times with SSC washing
buffer. After blocking with 10% donkey serum, samples were subjected to IF staining and visualized
with Nikon A1R+confocal microscopy.

Real-time quantitative PCR
Total RNA was extracted with standard TRIzol methods. cDNA was synthesized using 5× All-In-
one RT Mastermix (Abm, Vancouver, Canada) and analyzed on QuantStudio1 qPCR system using
specific primer pairs listed in Supplementary Table 3. The relative expression level was calculated and
normalized to endogenous Gapdh.

Transcriptome analysis
For transcriptome RNA-seq analysis, crypts were isolated from Snora61+/+ and Snora61−/− mice
aged 8 weeks. The isolated crypts were seeded to generate organoids using the aforementioned
method. Approximately 12 hours later, the cultures were harvested and centrifuged to remove
contaminating cells, yielding primary organoids. Then total RNA was extracted with standard TRIzol
methods and subjected to RNA sequencing by Beijing Genomics Institute. Subsequent data analysis
was conducted by R studio.

Flow cytometry
For flow cytometric analysis, crypts were isolated from Snora61+/+ and Snora61−/− mice aged 8
weeks and digested into single cells by TrypLE Express (gibco, Cat# 12605). EpCAM+LGR5+ cells
were analyzed with FACS Aria IIIu instrument (BD Biosciences). Data was collected with FACS Aria
IIIu instrument and analyzed by FlowJo Version 10.8.1 software.

Dot blot
DNA fragments corresponding to different regions of the Lgr5 promoter were amplified by PCR
and purified via gel extraction (primers are listed in Supplementary Table 4). 1μg DNA sample was
mixed with 20×SSC at a 2:1 volume ratio, followed by denaturation at 95°C for 10 minutes and
immediately placed on ice. The annealed products were spotted onto nylon membranes at consistent
intervals and subjected to UV cross-linking. Unbound DNA was removed by washing the membranes
with 2×SSC. Prehybridization of the membranes was performed using hybridization buffer at 65°C for
2 hours. Subsequently, the Snora61 probe was denatured and added to the hybridization solution,
followed by overnight incubation at 65°C. After stringent washing, the membranes were blocked,
incubated with secondary antibodies, and chemiluminescent signals were detected.

ChIRP-seq and ChIRP-qPCR
Crypts isolated from WT mice aged 8 weeks were digested into single cells. ISCs were isolated
by FACS and cross-linked with 1% formaldehyde at 37°C for 10 min and quenched by 0.1 M glycine
for 5 min. Then cells were washed twice with PBS, lysed with Lysis buffer (1% SDS, 10 mM EDTA, 50
mM Tris, supplemented with PMSF, proteinase inhibitor cocktails and M5 RNase inhibitor) and
sonicated to produce 200~500 bp DNA fragments. Lysates were incubated with biotinylated antisense
probes against Snora61 or scramble probe for 4 hours at 37°C. Streptavidin magnetic beads were
then added to isolate probe-binding complexes. After washing, DNAs were extracted from beads.
Subsequent DNA library construction, ChIRP-seq, and data analysis were performed by Novogene
Co., Ltd. (Beijing). The analysis results were further validated by ChIRP-qPCR using primer pairs listed
in Supplementary Table 3.

ChIP assay
Crypts isolated from WT mice aged 8 weeks were digested into single cells and cross-linked with
1% formaldehyde at 37°C for 10 min and quenched by 0.1 M glycine for 5 min. Then cells were washed
twice with PBS, lysed with Lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, supplemented with PMSF,
proteinase inhibitor cocktails) and sonicated to produce 200~500 bp DNA fragments. Lysates were
pre-cleared with Protein A/G magnetic beads (MCE, Cat# HY-K0202) and then incubated with 4 μg
indicated antibodies overnight at 4°C. Protein A/G magnetic beads were then added to isolate
antibody-binding complex. After washing steps, DNA were extracted from beads and analyzed by
qPCR using primer pairs listed in Supplementary Table 3.

Dual luciferase reporter assay
For luciferase reporter assay, the full-length promoter of Lgr5 (from −2000 to 0 upstream from
transcriptional start site) or truncated Lgr5 promoter was cloned into pGL3 vector. Snora61 sequence
was constructed into pLVX-IRES-Puro vector for overexpression. MC38 cells were seeded in 24-well
plate one day before transfection. For transfection of each well, 100 ng pGL3 plasmid, 1 ng pRL-TK
and 500 ng pLVX-IRES-Puro plasmid were used. Luminescence signals were analyzed using Dual
Luciferase Reporter Gene Assay Kit (Beyotime, Cat# RG027) according to the manufacturer’s protocol.

Nuclear run-on assay
Crypts were isolated from Snora61+/+ and Snora61−/− mice aged 8 weeks and digested into single
cells by TrypLE Express. Cells were suspended in nuclear extraction buffer (10 mM Tris-HCl, 150 mM
KCl, 4mM MgOAc, pH 7.4), followed by centrifugation to collect cell nuclei pellets. Pellets were lysed
by lysis buffer (nuclear extraction buffer supplemented with 0.5% NP-40), followed by sucrose density
gradient centrifugation to prepare transcriptional active crude nuclei components. Crude nuclei were
incubated with biotin labeling mix and RNase inhibitor at 28°C for 5 min. RNAs were extracted using
Trizol reagent according to the manufacturer’ s instructions. DNA was digested by DNase I for 15 min
at room temperature. RNA transcripts were enriched with streptavidin affinity beads, followed by
reverse transcription and RT-PCR analysis.

Chromatin accessibility assay
For chromatin accessibility assay, crypts were isolated from Snora61+/+ and Snora61−/− mice
aged 8 weeks and digested into single cells by TrypLE Express. Cells were suspended with Lysis
buffer (60 mM KCl, 10 mM HEPES, 1 mM EDTA, 1 mM DTT, 0.075% NP-40 and 1 mM PMSF, pH
7.6) to remove cytoplasmic components. Nuclei pellets were resuspended with DNase reaction buffer
(1 mM EDTA, 0.1 mM EGTA, 1 mM MgCl2, 0.5 mM CaCl2 and 5% Sucrose), added 0 U or 2 U DNase
I and incubated at 37°C for 5 min, followed by genomic DNA extraction and qPCR analysis of
indicated chromatin regions.

Nuclear and cytoplasmic fraction isolation
Cytoplasmic and nuclear fraction were extracted using NE-PER Nuclear and cytoplasmic
extraction according to the manufacturer’s protocol. Briefly, the cells were suspended and lysed with
cytoplasmic extraction buffer and then centrifuged to separate the nuclear fraction from the
cytoplasmic fraction. Subsequently, the nuclear fraction was lysed with nuclear extraction buffer.

Nucleoplasm and nucleolus fraction isolation
Break nuclei by sonicating the nuclear fraction on ice using several bursts of 10 sec at 400W at
10s intervals for 10 times. After each burst, verify the appearance of the suspension by examining a
drop of the suspension by phase contrast microscopy. Stop the sonication when intact nuclei cannot
be detected in the suspension. Very gently add 880 mM sucrose under the sonicated nuclei with a
micropipettor and then centrifuge 20 min at 2000 g, 4°C. After centrifugation, collect the supernatant,
which contains the nucleoplasmic fraction. Resuspend the purified nucleolar fraction in a minimal
volume of 340 mM sucrose buffer and check the quality of purification by visualizing under a phase
contrast microscope.

RNA pull-down assay
For RNA pull-down assay, 1×105 crypts were isolated from WT mice aged 8 weeks. Crypts cell
nuclear fraction was collected using NE-PER Nuclear and cytoplasmic extraction as previously
described and lysed with RIPA lysis buffer (1% Deoxycholate sodium, 150 mM NaCl, 50 mM Tris-
HCl, 0.1% SDS and 1% Triton X-100, pH 7.2). Supernatants were incubated with 2 μg biotin-labeled
Snora61 or antisense control probe overnight at 4°C and incubated with 60 μl Streptavidin Magnetic
Beads (Beyotime) for 4 h. Then precipitated components were separated with SDS-PAGE and silver
staining. For pull‑down assay shown in Figure 4a, the two most prominent differential bands (located
at approximately 42 kDa and 30 kDa) were cut for mass spectrometry analysis. Candidate proteins
identified by mass spectrometry for these two bands are presented in Supplementary Table 6.

Co-immunoprecipitation (Co-IP) assay
For Co-IP assay, 1×105 crypts were isolated from WT mice aged 8 weeks. Crypts cells nuclear
fraction was collected using NE-PER Nuclear and cytoplasmic extraction and lysed with RIPA lysis
buffer. Supernatants were incubated with anti-RBMX antibody (1:50) or IgG isotype. Then
precipitated components were separated with 12% SDS-PAGE electrophoresis, followed by silver
staining. For co-IP assay shown in Figure 5a, the band of RBMX at approximately 42 kDa and the
most prominently differential band around 24 kDa was cut for mass spectrometry analysis. Candidate
proteins identified by mass spectrometry for these bands are presented in Supplementary Table 7.

Statistical and reproducibility
For statistical evaluation, an unpaired Student’s t-test was applied for calculating statistical
probabilities in this study. For all panels, at least three independent experiments were performed and
representative experiments are shown. Data were analyzed by using Microsoft Excel or GraphPad
version 9. P-values smaller than 0.05 were regarded as statistical significance.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Jiacheng He and colleagues identify Snora61 as a novel regulator of intestinal stem cell (ISC) self-renewal and intestinal regeneration. The authors provide evidence that Snora61 exerts its function through the transcriptional regulation of Lgr5, mediated by the RNA-binding proteins Rbmx and Hmgb2. While this newly proposed regulatory mechanism is intriguing and potentially significant, several aspects of the study should be revisited to better generalize and consolidate the findings.\n- It remains unclear whether the role of Snora61 in regulating Lgr5 expression is specific to the mouse system or if it also applies to human intestinal stem cells. Clarifying this point is essential to establish the broader relevance of this regulatory mechanism beyond a mouse genetic background. The authors should assess the impact of Snora61 silencing on LGR5 expression and clonogenic potential in wild-type human intestinal organoids. If Snora61 is relevant in human, silencing Snora61 in human APC mutant ISC organoid should have little to no impact, since the wnt pathway is activated downstream of lgr5.\n- The authors performed RNA-seq to assess the impact of Snora61 knockout on gene expression. However, the analysis was limited to a subset of stemness-related genes, which overlooks the broader insights that an unbiased global analysis could provide. To strengthen the manuscript, the authors should present a heatmap of the top up- and down-regulated genes and perform a gene set enrichment analysis to identify signatures, significantly affected biological processes and pathways. I would expect that Wnt target genes and perhaps other programs are deregulated.\n- In figure 3G, the authors demonstrated that Snora61 is able to physically bind a region in the promoter of Lgr5, using ChIRP. In line with the first comment, the authors should perform ChIRP-seq, to present the global DNA binding capability of Snora61. This new dataset could then be intersected with the RNAseq analysis to determine all potential direct targets of Snora61. In addition, ChIP-seq or cut-n-Run of RBMX and HMGB2 should be performed to further demonstrate the extensive or limited collaboration between Snora61-RBMX-HMGB2 at the transcriptional level.\n- It remains unclear how Snora61-RBMX-HMGB2 find their target(s). Is Snora61 associating to DNA by complementarity? Do the binding of RBMX and HMGB2 to Lgr5 promoter partly depend on RNA? Is Snora61 facilitating the binding between RBMX and HMGB2? The fact that silencing either RBMX and HMGB2 in the Snora61 KO background further decreases Lgr5 expression suggests a role independent from Snora61 or that snora61 fine tunes optimal binding\\regulation. Few experiments should be done to clarify these questions.\n- In figure 1D, the authors are presenting the expression of Snora61 by RNA ISH. Since Snora61 is mainly express in SI and spleen, it would be good to complement the panel with spleen expression as well.\n- In figure 2, would be great to complement with the analysis of heterozygous animal. Any impact on SI length?, villi structure? That could help to point out toward a potential dosage dependent function.\n- Ensure that Snora61 expression data (qPCR) is presented in each knockdown/overexpression experiments.\n- In figure 4A and 5A, other bands appear specific (35 kDa\\4A and 55 kDa\\5A). What is the identity of these proteins? Any possible role in the function of Snora61?\n- Western blot quantifications should be added.\nNicolas Leveille", "reviewer_2": "This study presents a role of Snora61 in intestinal stem cell (ISC) self-renewal. The authors elucidate a novel regulatory axis —Snora61 interacting with Rbmx to recruit Hmgb2, thereby promoting Lgr5 transcription. This manuscript makes a significant contribution to the understanding of ISC regulation and snoRNA biology. However, given that Snora61 likely participates in multiple regulatory pathways as Snora61 knockout still impairs stemness in Lgr5-deficient crypts, it remains uncertain from the current data whether the observed effects on stemness are exclusively mediated through Lgr5. Moreover, additional experiments are warranted to clarify the Snora61-Rbmx-Hmgb2 axis in ISC maintenance. More specifically:\n1. Figure 1: The in situ hybridization images should be enlarged. The colocalization between Snora61 and Lgr5 is not clearly demonstrated. Additional colocalization analyses with other cell populations—such as +4 cells, Paneth cells, and differentiated cells—are needed.\n2. RNA-seq results: The change in Lgr5 expression is less than two fold and may not be significant. A comprehensive analysis of all differentially expressed genes is required, including upregulated and downregulated pathways (e.g., KEGG, GO) to assess the broader transcriptional impact of Snora61 knockout.\n3. A genome-wide assessment of Snora61-bound DNA regions via CHIRP-seq or array is necessary. These data should be integrated with RNA-seq results to identify direct Snora61 targets.\n4. DNA-binding site mutant: It is important to test whether the DNA-binding-deficient mutant of Snora61 can rescue organoid stemness, to validate the functional relevance of the promoter interaction.\n5. Figure 4: The authors should include overexpression of an Rbmx mutant lacking the Snora61-binding domain. Assessing stemness and Lgr5 expression in this context would clarify whether Rbmx function depends on Snora61 binding.\n6. Figure 5: There is inconsistency in interpretation. Panel K shows that Hmgb2 binding to the Lgr5 promoter depends on Snora61. However, Hmgb2 knockdown still affects stemness and Lgr5 expression in Snora61-deficient cells. This contradiction requires clarification.\n7. Figure 5 (panels F–L): These experiments should be repeated in Rbmx-knockout ISCs to determine whether the regulatory effects of Hmgb2 on Lgr5 expression and stemness are Rbmx-dependent.\n8. Figure 6: It is unclear how the authors generated Lgr5 knockout mice, given that previous studies have reported 100% neonatal lethality in Lgr5-null models (Mol Cell Biol. 2004 Nov;24(22):9736–9743.). Clarification on the viability, developmental stage, and genetic background of these knockout animals is needed. The interpretation also appears inconsistent. If Snora61’s function is Lgr5-dependent, why does additional Snora61 deletion still have an effect in Lgr5-deficient mice?", "reviewer_3": "In this manuscript, the authors propose a role for the ISC-specific expressed snoRNA Snora61 to modulate Lgr5 gene expression and impair intestinal regeneration. The authors demonstrated that Snora61 regulates the intestinal stem cell marker Lgr5 via RBMX to recruit HMGB2. However, some data are unconvincing, and some are inconsistent with each other. In addition, key issues remain unsolved.\nMajor points:\n1. As Lgr5⁺ ISCs are the principal stem cell population in both the small intestine and colon. Given the absence of Snora61 expression in the colonic epithelium (Fig 1C), Lgr5 expression is regulated with very different mechanisms in the colon? Also, how to explain the differential expression pattern of Snora61 between the small and large intestinal epithelia?\n2. Fig 1E: The in situ hybridization showed that Snora61 is also highly expressed in mesenchymal cells in villi. How do the authors rule out a potential functional role for Snora61 in mesenchymal cells, independent of its role in epithelial stem cells?\n3. Fig 2: It is recommended that the authors perform RNA-seq and subsequent bioinformatic analyses on Snora61 knockout mouse crypts under both homeostatic and post-irradiation conditions. Further functional enrichment analyses (e.g., GO, KEGG, GSEA) would help elucidate the molecular basis underlying the observed phenotypes.\n4. Fig 2E: It is important to include qPCR or Northern blot data demonstrating whether Snora61 expression is upregulated following irradiation. Are regenerative genes downregulated upon Snora61 deletion?\n5. If Snora61 only controls Lgr5 expression as this study shows, and its expression pattern is minimally overlapped with Ki67, how does Snora61 KO impair Ki67 expression and proliferation of Ki67+ TA cells?\n6. The data show in Fig. 3A-B are convincing and not well analyzed. Genes such as Tert, Ascl2, Tnfrsf19 and Bmi1 exhibited differing expression patterns between RNA-seq and Q-PCR. How to reconcile the discrepancy? The authors should re-analyzed the RNA-seq data to assess potential effects on other cell lineage or pathways. A Log2FC of 0.8 is not particular robust, warranting a broader examination.\n7. Fig. 3C: Lgr5 is still expressed at a large amount in Snora61 knockout (only about 50% reduction). Does a half decrease of Lgr5 levels contribute to all the phenotypes described in this study?\n8. Fig 3J-3L: Providing visualization of different peaks (e.g., via IGV software) would significantly strengthen the findings.\n9. Fig 4F and 4G: RBMX expression in tissues is primarily localized to the crypt base in mice, however, it appears in the nuclei of nearly all cells in the small intestinal organoids. How to explain?\n10. Fig 4H-4I: Given that RBMX expression is significantly broader than Snora61, it is difficult to attribute the observed effects specifically to a “Snora61-RBMX” interaction. Similar problems also occur in Fig 5E. The current conclusion is over-stated (Page 7, line 173).\n11. Fig 5: Immunofluorescence staining should be performed to determine Hmgb2 localization in the small intestine tissue. Moreover, it is important to show co-localization of HMGB2 and Snora61.\n12. The authors should test whether Hmgb2 overexpression can rescue the phenotypes caused by Snora61 knockout, including decreased Lgr5 expression.\n13. Fig 6 is problematic. Why to knock down Lgr5 in the context of Snora61 deletion? The worse phenotype observed in Snora61-/- + sgLgr5 is unsurprising, as Snora61 deletion already impairs ISCs. Also, If Snora61 functions primarily through Lgr5, why do Snora61+/+ + sgLgr5 and Snora61-/- + sgLgr5 groups still showed different, even after Lgr5 knockdown in both? The authors provide an explanation for this observation. Furthermore, dFz7-21 treatment cannot address these issues.\n14. Fig. 6C: it is surprised to see that Lgr5 KD reduced the crypt number to 50%, similar to Snora61 KO, but Snora61 KO only decreases about 50% Lgr5 expression.\n15. Does Snora61 also control Lgr5 expression in human intestine?\n16. Key information is missing: Regarding the mouse models subjected to gene knockout/irradiation, there are no data on: (a)mortality, (b) body weight dynamics, (c) DAI score, and (d) the temporal pattern of Lgr5 reduction (i.e., whether it is sustained or reversible).\n17. No information of the time points for tissue sample collection in all mouse-related experiments. Similarly, no specification of the time points for organoids culture and image acquisition throughout the manuscript.\nOther points:\n1. Fig 1D: The gene name Olfm4 should be italicized to comply with standard gene nomenclature conventions.\n2. Gene names such as Lgr5, Hmgb2, and Oct4 should be italicized when referring to mouse genes, whereas the corresponding protein names should be presented in all capital letters. Please go through the manuscript carefully.\n3. Page 3, line 47: There is a misstatement regarding snoRNA, which should be defined as small nucleolar RNA.\n4. Page 4, line 78: The main text mentions “microarray”, but the legend of Figure 1B is actually labeled as RT-qPCR.\n5. Latin terms such as in situ should be italicized throughout the manuscript.\n6. It is suggested that Figure 1I be moved to the supplementary materials to improve the flow and focus of the main figures.", "reviewer_4": "This paper describes that Snora61 is highly expressed in intestinal stem cells (ISCs). The absence of Snora61 significantly impaired ISC self-renewal and intestinal regenerative capacity. Snora61 bound to Rbmx, which then recruited the co-transcription factor Hmgb2 to the promoter region of the Lgr5 gene. This promoted transcription of the Lgr5 gene, which was crucial for stem cell self-renewal. The authors demonstrated that a new regulatory network involving Snora61-Rbmx-Hmgb2-Lgr5 contributes to ISC maintenance. Overall, this study provides important insights into ISC self-renewal. However, several unresolved issues remain that need to be clarified.\nLine 70\nWhy was analysis using snoRNA microarrays necessary? The authors should clarify their motivation. Furthermore, the expression of snora61 in IEC is only twice that in ISC (Figure 1A). It is difficult to consider this significant. Further research should be conducted by comparing it in detail with other snoRNAs. The authors should include a table in the supplementary materials showing the expression levels of other snoRNAs to make this information clear to readers.\nLine 91\nThe Snora61 genes in humans and mice, designated NR_002987.1 and NR_034046.1, respectively, exhibit significant sequence differences. The authors should clarify whether they knocked out NR_034046.1 in mice or whether they knocked out another Snora61 homologous gene. This point should be explained in detail here, and further information should be added to the text.\nLine 130\nAs shown in Figure 3A, the ratio of Lgr5 expression levels between Snora61+/+ and Snora61-/- mice is approximately twofold at most. Therefore, it is not necessary to proceed with Lgr5 as the primary target. To thoroughly discuss this with readers, it is necessary to present RNA-seq results confirming this finding in a table as supplementary material.\nLine 156\nThe experimental methods are inadequately described. For this pull-down, the authors should specify the amounts of biotin-labeled snora61 and crypt cells used. In Figure 4A, which shows the experimental results, various different bands are observed in the experimental and control groups. Many proteins other than Rbmx must have bound.The authors should present a table in the supplementary materials listing all identified proteins. Additionally, they should provide detailed explanations regarding the criteria used to select Rbmx from among these proteins.\nLine 178\nFigure 5A is very strange. Hmgb2 is pulled down and visible as a band, but the band of Rbmx, which should be co-precipitated, is not visible. Based on the principle of this experiment, Rbmx should precipitate more than Hmgb2 because immunoprecipitation is performed with an antibody against Rbmx. The authors need to explain why Hmgb2 precipitates in large amounts while Rbmx does not."}, "author_response": "Point-by-point response to the reviewers\nReviewer #1:\nIn this manuscript, Jiacheng He and colleagues identify Snora61 as a novel regulator\nof intestinal stem cell (ISC) self-renewal and intestinal regeneration. The authors provide\nevidence that Snora61 exerts its function through the transcriptional regulation of Lgr5,\nmediated by the RNA-binding proteins RBMX and HMGB2. While this newly proposed\nregulatory mechanism is intriguing and potentially significant, several aspects of the study\nshould be revisited to better generalize and consolidate the findings.\n1. It remains unclear whether the role of Snora61 in regulating Lgr5 expression is specific\nto the mouse system or if it also applies to human intestinal stem cells. Clarifying this point\nis essential to establish the broader relevance of this regulatory mechanism beyond a\nmouse genetic background. The authors should assess the impact of SNORA61 silencing\non LGR5 expression and clonogenic potential in wild-type human intestinal organoids. If\nSnora61 is relevant in human, silencing SNORA61 in human APC mutant ISC organoid\nshould have little to no impact, since the Wnt pathway is activated downstream of LGR5.\nAnswer: This is a good point. We added genomic information on human SNORA61\nand its homology with mice in the new Supplementary Figure 1c. Snora61 and SNORA61\nshare similar genomic characteristics: they are each encoded within the SNHG (Small\nNucleolar RNA Host Gene) family gene Snhg12/SNHG12 as two of their eight snoRNA\nproducts, and exhibit 73.68% sequence homology (new Supplementary Figure. 1d). We\ndesigned shRNAs targeting SNORA61 and validated their knockdown efficiency.\nKnockdown of SNORA61 inhibited growth of human normal intestinal organoids and\nreduced LGR5 expression (new Supplementary Figure. 1e), but did not affect the growth\nof human intestinal organoids with APC mutations (new Supplementary Figure. 7i).\n2. The authors performed RNA-seq to assess the impact of Snora61 knockout on gene\nexpression. However, the analysis was limited to a subset of stemness-related genes, which overlooks the broader insights that an unbiased global analysis could provide. To\nstrengthen the manuscript, the authors should present a heatmap of the top up- and down-\nregulated genes and perform a gene set enrichment analysis to identify signatures,\nsignificantly affected biological processes and pathways. I would expect that Wnt target\ngenes and perhaps other programs are deregulated.\nAnswer: We reanalyzed the RNA-seq data in the new Figure 3. GO analysis revealed\nthat downregulated genes with Snora61 knockout were enriched in pathways such as gene\ntranscription, cell proliferation/differentiation, and digestive tract development (new Figure.\n3a). Further analysis of stemness-related genes indicated that Lgr5 exhibited the most\npronounced downregulation (new Figure. 3b). In addition, we added RNA-seq of ISCs from\nSnora61+/+ and Snora61/ mice post irradiation. According to the RNA-seq results, a set\nof downstream target genes of LGR5 also showed downregulation, and these differences\nbecame more pronounced during the repair process following irradiation-induced injury\n(new Figure. 3c, d). Consequently, the gene expression profile in Snora61/ ISCs showed\nsignificantly reduced enrichment in Wnt signaling pathways post irradiation (new Figure.\n3e). Therefore, in this study we focused on the mechanistic investigation of the transcription\nactivation of Lgr5 gene in the regulation of ISCs.\n3. In figure 3G, the authors demonstrated that Snora61 is able to physically bind a region\nin the promoter of Lgr5, using ChIRP. In line with the first comment, the authors should\nperform ChIRP-seq, to present the global DNA binding capability of Snora61. This new\ndataset could then be intersected with the RNA-seq analysis to determine all potential\ndirect targets of Snora61. In addition, ChIP-seq or cut-n-Run of RBMX and HMGB2 should\nbe performed to further demonstrate the extensive or limited collaboration between\nSnora61-RBMX-HMGB2 at the transcriptional level.\nAnswer: This is a very good point. We tried to perform ChIRP-seq and ChIP-seq\nassays several times. However, because the commercial antibodies had not good quality\nfor ChIP-seq assays, we failed this assay. On the other hand, possibly due to inadequate\nquantity of isolated intestinal stem cells, library construction of ChIRP-seq was\nunsuccessful at the sequencing company. As an alternative assay, we added RBMX ChIP\nexperiments in the new Figure 4, demonstrating that RBMX was enriched at the promoter\nregion of Lgr5 (new Figure. 4k, l), thereby strengthening our conclusions.\n4. It remains unclear how Snora61-RBMX-HMGB2 find their target(s). Is Snora61\nassociating to DNA by complementarity? Does the binding of RBMX and HMGB2 to Lgr5\npromoter partly depend on RNA? Is Snora61 facilitating the binding between RBMX and\nHMGB2? The fact that silencing either RBMX and HMGB2 in the Snora61 KO background\nfurther decreases Lgr5 expression suggests a role independent from Snora61 or that\nSnora61 fine tunes optimal binding\\regulation. Few experiments should be done to clarify\nthese questions.\nAnswer: This is a good suggestion. Snora61bound to the promoter region\nofLgr5through complementary base pairing (new Figure. 3i) and recruited HMGB2 after\nassociating with RBMX. Snora61 knockout abolished the enrichment of RBMX and\nHMGB2 at theLgr5promoter (new Figure. 4l and Figure. 5m). Of note,Snora61did not\ninfluence the direct interaction between RBMX and HMGB2 (new Figure. 5d). In fact,\nsilencing either RBMX and HMGB2 in the Snora61 KO further decreased Lgr5 expression,\nsuggesting Lgr5expression was also regulated by Snora61-independent mechanisms,\nwhich is beyond the scope of this current study.\n5. In figure 1D, the authors are presenting the expression of Snora61 by RNA ISH. Since\nSnora61 is mainly express in SI and spleen, it would be good to complement the panel\nwith spleen expression as well.\nAnswer: We provided a staining panel with spleen expression (new Figure. 1d).\n6. In figure 2, would be great to complement with the analysis of heterozygous animal. Any\nimpact on SI length? villi structure? That could help to point out toward a potential dosage\ndependent function.\nAnswer: We added phenotypic analysis of heterozygous mice in the new\nSupplementary Figure 2k-m. The expression level ofSnora61 in Snora61+/ mice was\napproximately half of that in Snora61+/+ mice (new Supplementary Fig. 2j). We observed\nanalogous but less severe stemness suppression in Snora61+/ mice compared to\nSnora61/ mice, including shortened small intestine and villus length, reduced crypt\nnumber, and impaired organoid formation efficiency (new Supplementary Fig. 2k-m).\nThese results suggest that Snora61 function may exhibit a dose-dependent effect.\n7. Ensure that Snora61 expression data (qPCR) is presented in each\nknockdown/overexpression experiments.\nAnswer: We added RT-qPCR validation of the knockdown/overexpression efficiency\nof Snora61 (new Supplementary Figure. 1a and Supplementary Figure. 2e).\n8. In figure 4A and 5A, other bands appear specific (35 kDa\\4A and 55 kDa\\5A). What is\nthe identity of these proteins? Any possible role in the function of Snora61?\nAnswer: In Figure 4A, the band observed at approximately 35 kDa primarily\ncorresponded to heterogeneous nuclear ribonucleoproteins (hnRNPs) involved in RNA\nsplicing (Attached Figure. 1a). Since these proteins commonly contained RNA recognition\nmotifs (RRMs), they often produced non-specific binding signals in pulldown assays, as\nevidenced by the presence of this band in both the sense and scrambled control groups.\nTherefore, these candidates were excluded to further considerate.\nIn Figure 5A, the bands above 55 kDa are primarily attributed to keratins, which were\ncommon cytoskeletal proteins in epithelial cells (Attached Figure. 1b). These proteins were\nprimarily localized in the cytoplasm, which was inconsistent with the nuclear localization of\nRBMX, and were therefore excluded.\n9. Western blot quantifications should be added.\nAnswer: We provided all Western blot experiments requiring comparative analysis\nwith corresponding quantitative graphs (new Supplementary Fig. 3h, Supplementary Fig.\n4f, Supplementary Fig. 5c, Supplementary Fig. 7a and Supplementary Fig. 7e).\nReviewer #2:\nThis study presents a role of Snora61 in intestinal stem cell (ISC) self-renewal. The\nauthors elucidate a novel regulatory axis—Snora61 interacting with RBMX to recruit\nHMGB2, thereby promoting Lgr5 transcription. This manuscript makes a significant\ncontribution to the understanding of ISC regulation and snoRNA biology. However, given\nthat Snora61 likely participates in multiple regulatory pathways as Snora61 knockout still\nimpairs stemness in LGR5-deficient crypts, it remains uncertain from the current data\nwhether the observed effects on stemness are exclusively mediated through LGR5.\nMoreover, additional experiments are warranted to clarify the Snora61-RBMX-HMGB2 axis\nin ISC maintenance. More specifically:\n1. Figure 1: The in-situ hybridization images should be enlarged. The colocalization\nbetween Snora61 and LGR5 is not clearly demonstrated. Additional colocalization\nanalyses with other cell populations—such as +4 cells, Paneth cells, and differentiated\ncells—are needed.\nAnswer: We added a FISH staining image of the intestinal crypt in the new Figure. 1h.\nThe image clearly showed that Snora61 was primarily localized at the crypt base,\ndemonstrating strong co-localization with LGR5+ intestinal stem cells. Additionally,\nSnora61 was also expressed at low levels in Paneth cells (arranged in an alternating\npattern with intestinal stem cells at the crypt base).\n2. RNA-seq results: The change in Lgr5 expression is less than two-fold and may not be\nsignificant. A comprehensive analysis of all differentially expressed genes is required,\nincluding upregulated and downregulated pathways (e.g., KEGG, GO) to assess the\nbroader transcriptional impact of Snora61 knockout.\nAnswer: This is a good point. Our reanalysis of the RNA-seq data, presented in the\nnew Figure 3, showing that GO analysis identified enrichment of downregulated genes\nfollowing Snora61 knockout in pathways including gene transcription, cell\nproliferation/differentiation, and digestive tract development (new Figure. 3a). Assessment\nof stemness-related genes further revealed that Lgr5 was the most significantly\ndownregulated among them (new Figure. 3b). Additionally, we performed RNA-seq on\nISCs from both Snora61+/+ and Snora61/ mice post irradiation. The results demonstrated\nthat a group of downstream target genes of LGR5 were also downregulated to various\nextents, with these differences becoming more pronounced during the repair phase after\nirradiation-induced damage (new Figure. 3c, d). Accordingly, post-irradiation gene\nexpression profiles in Snora61/ ISCs exhibited markedly reduced enrichment for the Wnt\nsignaling pathway (new Figure. 3e). In light of these findings, we focused on our\nmechanistic studies toward understanding the transcriptional activation of Lgr5.\n3. A genome-wide assessment of Snora61-bound DNA regions via ChIRP-seq or array is\nnecessary. These data should be integrated with RNA-seq results to identify direct Snora61\ntargets.\nAnswer: This is a very good point. We tried to perform ChIRP-seq assays several\ntimes. However, possibly due to inadequate quantity of isolated intestinal stem cells, library\nconstruction of ChIRP-seq was unsuccessful at the sequencing company.\n4. DNA-binding site mutant: It is important to test whether the DNA-binding-deficient mutant\nof Snora61 can rescue organoid stemness, to validate the functional relevance of the\npromoter interaction.\nAnswer: We added the corresponding experimental data in the new Figure. 3q and\nSupplementary Figure. 3l. As expected, mutant Snora61 could not restore the formation\nefficiency in Snora61/ organoids (new Fig. 3q and Supplementary Fig. 3l).\n5. Figure 4: The authors should include overexpression of an RBMX mutant lacking the\nSnora61-binding domain. Assessing stemness and Lgr5 expression in this context would\nclarify whether RBMX function depends on Snora61 binding.\nAnswer: We added the corresponding experimental data in the new Figure. 4j and\nSupplementary Figure. 4h. To assess the functional requirement of the RBMX-Snora61\ncomplex, we performed rescue experiments inshRbmx organoids. Expression of an RRM-\ndeletion mutant of RBMX failed to rescue the impaired organoid formation with\nlowLgr5expression, unlike the full-length protein (new Fig. 4j and Supplementary Fig. 4h).\n6. Figure 5: There is inconsistency in interpretation. Panel K shows that HMGB2 binding\nto the Lgr5 promoter depends on Snora61. However, Hmgb2 knockdown still affects\nstemness and Lgr5 expression in Snora61-deficient cells. This contradiction requires\nclarification.\nAnswer: In this study, we focused exclusively on the cooperative function of HMGB2\nwith Snora61 to modulate Lgr5 transcription. Beyond its involvement in transcriptional\nregulation, HMGB2 may play other roles in chromatin remodeling and accessibility.\nTherefore, Hmgb2 knockdown may further affectLgr5expression by Snora61-\nindependent mechanisms inSnora61-knockout mice, which is beyond the scope of this\ncurrent study.\n7. Figure 5 (panels F–L): These experiments should be repeated in Rbmx-knockout ISCs\nto determine whether the regulatory effects of HMGB2 on Lgr5 expression and stemness\nare RBMX-dependent.\nAnswer: This is a good suggestion. We added the corresponding experimental data\nin the new Figure. 5n and Supplementary Figure. 6. To define RBMX's role in mediating\nHMGB2's function in ISCs, we generated AAV-mediated Rbmx conditional knockout mice\n(new Supplementary Fig. 6a). In this setting, Hmgb2 overexpression failed to rescue the\nimpaired organoid formation and reducedLgr5expression caused byHmgb2knockdown\n(new Supplementary Fig. 6b-d). Furthermore, Rbmx knockout abrogated the specific\nbinding of HMGB2 to theLgr5promoter (new Supplementary Fig. 6e). In addition, Rbmx\nknockdown inhibited the Lgr5 transcription activity in dual luciferase reporter assay (new\nFig. 5n). Altogether, we conclude that the regulatory effect of HMGB2 on Lgr5 expression\nand stemness are RBMX dependent.\n8. Figure 6: It is unclear how the authors generated Lgr5 knockout mice, given that previous\nstudies have reported 100% neonatal lethality in Lgr5-null models (Mol Cell Biol. 2004\nNov;24(22):9736–9743.). Clarification on the viability, developmental stage, and genetic\nbackground of these knockout animals is needed. The interpretation also appears\ninconsistent. If Snora61’s function is LGR5-dependent, why does additional Snora61\ndeletion still have an effect in Lgr5-deficient mice?\nAnswer: The methodology for generating the Lgr5 conditional knockout mice has\nbeen described in the Methods section: Lgr5-deletion mice were established using\nCRISPR/Cas9 approaches provided by Zhang’s lab. The sgRNAs were designed by online\nCRISPR design tool (https://crispor.gi.ucsc.edu/). The sgRNA sequences are listed in\nSupplementary Table 1. In brief, sgRNAs were cloned into pAAV-U6-gRNA-CBh-mCherry\n(Addgene, catalog 91947). AAV vectors were co-transfected with packaging plasmids\npHelper and pAnc80 into HEK293T cells. AAV were harvested at 72 h after transfection by\nrepeated freezing and thawing and filtered through 0.22 μm filters. AAV was injected into\nVillin-Cre;Cas9-KI mice aged 6 weeks via the tail vein. It can be confirmed that this\nknockout approach, at least within the experimental timeframe, did not cause lethality in\nthe mice.\nIt should be noted that the knockout strategy employed in Figure. 6 did not achieve\ncomplete ablation of Lgr5 expression, as evidenced by the Western blot and RT-qPCR\nresults (new Supplementary Fig. 7a, b). sgRNA operated at the genomic level to modulate\ngene expression, whereas Snora61 regulatedLgr5expression specifically at the\ntranscriptional stage. Therefore, under these conditions, Snora61 knockout could still exert\nan impact onLgr5expression, thereby further influencing the phenotypic outcomes in mice.\nOn the other hand, while our study revealed that Snora61 regulated ISC stemness by\nmodulating Lgr5 expression, Snora61 may also influence self-renewal of ISCs through\nadditional mechanisms, as suggested by the RNA-seq analysis (new Fig. 3a, b).\nReviewer #3:\nIn this manuscript, the authors propose a role for the ISC-specific expressed snoRNA\nSnora61 to modulate Lgr5 gene expression and impair intestinal regeneration. The authors\ndemonstrated that Snora61 regulates the intestinal stem cell marker LGR5 via RBMX to\nrecruit HMGB2. However, some data are unconvincing, and some are inconsistent with\neach other. In addition, key issues remain unsolved.\n1. As LGR5 ISCs are the principal stem cell population in both the small intestine and\ncolon. Given the absence of Snora61 expression in the colonic epithelium (Fig 1C), Lgr5\n⁺\nexpression is regulated with very different mechanisms in the colon? Also, how to explain\nthe differential expression pattern of Snora61 between the small and large intestinal\nepithelia?\nAnswer: This is a good point. Given the high expression of Snora61 in the small\nintestine (new Fig. 1e and Supplementary Fig. 1g), we thus focused on its function in the\nregulation of small intestinal stem cells. We acknowledge that distinct regulatory\nmechanisms may govern stem cell self-renewal in the colon, however, investigation of\nthese mechanisms is beyond the scope of this work.\nThe host gene encoding Snora61, Snhg12, belongs to the SNHG (Small Nucleolar\nRNA Host Gene) family, which is specialized in encoding snoRNAs. It has been well-\nestablished that SNHG expression itself exhibits considerable variation across different\norgans and tissues (Zhen N, Cell Mol Gastroenterol, 2023). Consequently, the RT-qPCR\nresults indicated that Snhg12 expression was significantly higher in small intestinal ISCs\ncompared to colonic ISCs (Attached Figure. 2a). The various expression levels ofSNHGs\nacross different organs arose from tissue-specific gene regulation, including epigenetic\nmechanisms, alternative splicing, andpost-transcriptional processing,which affected the\nproduction of both the host gene and its embeddedsnoRNAs (Zhen N, Cell Mol\nGastroenterol, 2023).\n2. Fig 1E: The in-situ hybridization showed that Snora61 is also highly expressed in\nmesenchymal cells in villi. How do the authors rule out a potential functional role for\nSnora61 in mesenchymal cells, independent of its role in epithelial stem cells?\nAnswer: This is a good point. Our phenotypic observations ofSnora61-knockout\nmice suggested that Snora61 influenced the maintenance of ISC stemness. Given the\npredominant expression of Snora61 in ISCs, we thus focused primarily on its regulatory\nrole in the regulation of ISCs. Furthermore, the functional impact of Snora61 and related\npathway proteins was validated through intestinal organoid formation assays. Since\nconventional intestinal organoid cultures lack mesenchymal components (Sato T, Nature\n2009), our observed phenotypes supported the conclusion that Snora61 functions\nindependently of mesenchymal cells in the regulation of ISC stemness.\n3. Fig 2: It is recommended that the authors perform RNA-seq and subsequent\nbioinformatic analyses on Snora61 knockout mouse crypts under both homeostatic and\npost-irradiation conditions. Further functional enrichment analyses (e.g., GO, KEGG,\nGSEA) would help elucidate the molecular basis underlying the observed phenotypes.\nAnswer: We supplemented the study with RNA-seq data following irradiation and\nperformed re-analysis of the RNA-seq results. GO analysis revealed that under\nhomeostatic conditions, genes downregulated uponSnora61knockout were enriched in\npathways such as gene transcription, cell proliferation/differentiation, and digestive tract\ndevelopment (new Figure. 3a). Further analysis of stemness-related genes\nidentifiedLgr5as the most significantly downregulated gene (new Figure. 3b). RT-qPCR\nvalidation confirmed various degrees of downregulation in a series ofLGR5downstream\ntarget genes, with these differences becoming more pronounced after irradiation (new\nFigure. 3c, d). Additionally, GSEA demonstrated that gene expression in Snora61/ISCs\npost irradiation showed significantly reduced enrichment in the Wnt signaling pathway (new\nFigure. 3e).\n4. Fig 2E: It is important to include qPCR or Northern blot data demonstrating whether\nSnora61 expression is upregulated following irradiation. Are regenerative genes\ndownregulated upon Snora61 deletion?\nAnswer: We added the dynamic expression profile of Snora61 during post-irradiation\ninjury repair in the new Supplementary Figure. 3k. We monitored the dynamic expression\nofLgr5andSnora61during intestinal repair post irradiation. Time-course analysis\nrevealed that after an initial synchronous decline withLgr5 due to stem cell\nloss,Snora61expression rebounded ahead ofLgr5recovery (new Supplementary Fig. 3k),\nindicating its specific upregulation within the remaining stem cell pool in response to\ndamage. This temporal pattern, wheresnora61expression increased independently of and\nprior toLgr5recovery, suggesting its role in facilitating the regenerative process by\nmodulatingLgr5.\nA set of regenerative genes showed various degrees of downregulation in Snora61/\nmice under homeostatic conditions, and these differences became more pronounced\nduring the repair process following irradiation-induced injury (new Figure. 3c, d).\n5. If Snora61 only controls Lgr5 expression as this study shows, and its expression pattern\nis minimally overlapped with Ki67, how does Snora61 KO impair Ki67 expression and\nproliferation of Ki67+ TA cells?\nAnswer: In this study, we showed that Snora61 regulated intestinal stem cell self-\nrenewal“primarily”, but not “exclusively”. On the other hand, Ki67 serves as a standard\nmarker for detecting cell proliferation. Our purpose in examining Ki67 was to assess\nchanges in proliferative capacity within the intestine and organoids resulting from\nalterations in intestinal stem cell stemness.\n6. The data show in Fig. 3A-B are convincing and not well analyzed. Genes such as Tert,\nAscl2, Tnfrsf19 and Bmi1 exhibited differing expression patterns between RNA-seq and Q-\nPCR. How to reconcile the discrepancy? The authors should re-analyze the RNA-seq data\nto assess potential effects on other cell lineage or pathways. A Log FC of 0.8 is not\n2\nparticular robust, warranting a broader examination.\nAnswer: During the preparation of RNA-seq samples, primary organoids were\nspecifically collected to minimize interference from non-target cells (detailed experimental\nprocedures have been updated in the Methods section). For the initial RT-qPCR validation,\nwe directly used total RNA isolated from mouse small intestinal crypts, which may account\nfor the minor inconsistencies observed in a subset of genes. To ensure consistency, we\nunified the sample sources for both assays (primary organoids) and updated the RT-qPCR\nresults in the new Supplementary Figure 3g. Both two analyses of stemness-related genes\nidentifiedLgr5as the most significantly downregulated gene (new Figure. 3b and\nSupplementary Figure. 3g).\nAs mentioned above, our RNA-seq analysis primarily focused on alterations in gene\nexpression within intestinal stem cells themselves. Following Snora61 knockout, Lgr5\nexhibited the most pronounced downregulation among stemness-associated genes (new\nFigure. 3b). This reduction in LGR5 levels led to the downregulation of a series of\ndownstream target genes of the Wnt pathway (new Figure. 3c). These transcriptional\ndifferences were further amplified during the injury repair process following irradiation (new\nFigure. 3d). Collectively, these findings provided a compelling explanation for the\nphenotypic observations in the knockout mice. Therefore, we focused on the mechanistic\ninvestigation of the transcriptional activation of Lgr5.\n7. Fig. 3C: Lgr5 is still expressed at a large amount in Snora61 knockout (only about 50%\nreduction). Does a half decrease of Lgr5 levels contribute to all the phenotypes described\nin this study?\nAnswer: LGR5 is a key receptor on intestinal stem cells and plays a critical role in\ntissue homeostasis, regeneration, and repair (Gehart H, Nat Rev Gastroenterol Hepatol,\n2021). LGR5 does not directly influence specific genes, rather, it acts as a signal amplifier\nthat extensively modulates downstream target gene expression indirectly through Wnt\npathway activation. Conversely, activated Wnt signaling can maintain or enhance LGR5\nexpression, establishing a positive feedback loop that stabilizes the stem cell state (de Lau\nW, Nature, 2011). Thus, even a 50% reduction in LGR5 expression may disrupt this\nactivation–expression homeostasis and substantially impair stemness. As mentioned\nabove, a set of downstream target genes of LGR5 also showed various degrees of\ndownregulation (new Figure. 3c). Furthermore, we focused on the mechanism by which\nSnora61 influenced intestinal stem cell stemness via Lgr5, Snora61 may also contribute\nto intestinal stem cell homeostasis through additional pathways.\n8. Fig 3J-3L: Providing visualization of different peaks (e.g., via IGV software) would\nsignificantly strengthen the findings.\nAnswer: The analysis of histone modification levels presented here was not based on\nsequencing data, but was achieved by using chromatin immunoprecipitation (ChIP)\nfollowed by quantitative PCR (qPCR). Therefore, the experimental results in the figure\nwere not presented in the form of peak graphs.\n9. Fig 4F and 4G: RBMX expression in tissues is primarily localized to the crypt base in\nmice, however, it appears in the nuclei of nearly all cells in the small intestinal organoids.\nHow to explain?\nAnswer: Due to limitations in commercially available antibodies and experimental\nconditions at the time, the initial fluorescence staining for RBMX in intestinal tissue sections\nwas incomplete. We purchased a validated antibody and optimized the protocol by\nincluding an antigen retrieval step. The repeated experiment, presented in the new\nSupplementary Figure. 4b, demonstrated that RBMX was localized throughout the\nintestinal epithelium.\n10. Fig 4H-4I: Given that RBMX expression is significantly broader than Snora61, it is\ndifficult to attribute the observed effects specifically to a “Snora61-RBMX” interaction.\nSimilar problems also occur in Fig 5E. The current conclusion is over-stated (Page 7, line\n173).\nAnswer: Although RBMX and HMGB2 exhibited broader expression patterns than\nSnora61 and lacked specificity to intestinal stem cells, here we focused on their\ncooperative role with Snora61 in regulatingLgr5expression, where Snora61 was enriched\nin the ISCs. RBMX and HMGB2 might have other roles in different settings, but in the ISCs\nSnora61 recruited the RBMX-HMGB2 complex to induce Lgr5expression for the regulation\nof ISC stemness.\n11. Fig 5: Immunofluorescence staining should be performed to determine HMGB2\nlocalization in the small intestine tissue. Moreover, it is important to show co-localization of\nHMGB2 and Snora61.\nAnswer: We added the corresponding experimental data in the new Figure. 5e.\nSnora61 and HMGB2 were co-localized in the nucleus of ISCs (new Fig. 5e).\n12. The authors should test whether Hmgb2 overexpression can rescue the phenotypes\ncaused by Snora61 knockout, including decreased Lgr5 expression.\nAnswer: We added the corresponding experimental data in the new Supplementary\nFigure. 5g, h. Overexpression of Hmgb2 failed to restore the impaired organoid formation\nandLgr5expression in Snora61/ organoids (new Supplementary Fig. 5g, h).\n13. Fig 6 is problematic. Why to knock down Lgr5 in the context of Snora61 deletion? The\nworse phenotype observed in Snora61/ + sgLgr5 is unsurprising, as Snora61 deletion\nalready impairs ISCs. Also, If Snora61 functions primarily through Lgr5, why do Snora61+/+\n+ sgLgr5 and Snora61/ + sgLgr5 groups still showed different, even after Lgr5 knockdown in\nboth? The authors provide an explanation for this observation. Furthermore, dFz7-21\ntreatment cannot address these issues.\nAnswer: This is a good suggestion. The knockout strategy used in Figure 6 did not\nresult in complete ablation of LGR5 expression, as demonstrated by Western blot and RT-\nqPCR analyses (new Supplementary Fig. 7a, b). While sgRNA functions at the genomic\nlevel to modulate gene expression, Snora61 specifically regulated Lgr5 at the\ntranscriptional stage. Consequently, even under these conditions, Snora61 knockout could\nstill affect Lgr5 expression (new Supplementary Fig. 7b), ultimately contributing to the\nphenotypic outcomes observed in mice. Furthermore, although our findings indicated that\nSnora61 regulated intestinal stem cell stemness through modulation of Lgr5 expression,\nRNA-seq data suggested that Snora61 might also affect intestinal stem cell self-renewal\nvia other mechanisms (new Fig. 3a, b). For these reasons, the double-knockout mice might\nbe expected to display a more severe phenotype than single-knockout models.\n14. Fig. 6C: it is surprised to see that Lgr5 KD reduced the crypt number to 50%, similar to\nSnora61 KO, but Snora61 KO only decreases about 50% Lgr5 expression.\nAnswer: These results suggested that the functions of Snora61 in intestinal stem cells\nare likely multifaceted. Snora61 regulates intestinal stem cell self-renewal“primarily”,\nthough not “exclusively”, through modulating Lgr5 expression.\n15. Does Snora61 also control Lgr5 expression in human intestine?\nAnswer: We added genomic information on human SNORA61 and its human-mouse\nhomology in the new Supplementary Figure 1. We designed shRNAs targeting SNORA61\nand validated their knockdown efficiency. Knockdown of SNORA61 inhibited growth of\nhuman normal intestinal organoids and reduced LGR5 expression (new Supplementary\nFigure. 1e).\n16. Key information is missing: Regarding the mouse models subjected to gene\nknockout/irradiation, there are no data on: (a)mortality, (b) body weight dynamics, (c) DAI\nscore, and (d) the temporal pattern of Lgr5 reduction (i.e., whether it is sustained or\nreversible).\nAnswer: We added the requested experimental data in the new Supplementary Figure.\n2h, i, Supplementary Figure. 3j and Supplementary Figure. 7g, h, with corresponding\ndescriptions added to the manuscript. Snora61 or Lgr5 knockout caused more severe body\nweight loss and a higher disease activity index (DAI) score. Of note, the experimental\nconditions employed in our study did not result in any mouse mortality, therefore, survival\nrates were not statistically evaluated.\n17. No information of the time points for tissue sample collection in all mouse-related\nexperiments. Similarly, no specification of the time points for organoids culture and image\nacquisition throughout the manuscript.\nAnswer: We thoroughly reviewed the manuscript and provided the relevant\nexperimental time points to the Methods section.\nOther points:\n1. Fig 1D: The gene name Olfm4 should be italicized to comply with standard gene\nnomenclature conventions.\nAnswer: We revised the relevant text accordingly.\n2. Gene names such as Lgr5, Hmgb2, and Oct4 should be italicized when referring to\nmouse genes, whereas the corresponding protein names should be presented in all capital\nletters. Please go through the manuscript carefully.\nAnswer: We thoroughly reviewed the manuscript and revised the relevant text\naccordingly.\n3. Page 3, line 47: There is a misstatement regarding snoRNA, which should be defined\nas small nucleolar RNA.\nAnswer: We revised this misstatement.\n4. Page 4, line 78: The main text mentions “microarray”, but the legend of Figure 1B is\nactually labeled as RT-qPCR.\nAnswer: We revised this error.\n5. Latin terms such as in situ should be italicized throughout the manuscript.\nAnswer: We revised the relevant text accordingly.\n6. It is suggested that Figure 1I be moved to the supplementary materials to improve the\nflow and focus of the main figures.\nAnswer: We changed this as suggested.\nReviewer #4:\nThis paper describes that Snora61 is highly expressed in intestinal stem cells (ISCs).\nThe absence of Snora61 significantly impaired ISC self-renewal and intestinal regenerative\ncapacity. Snora61 bound to RBMX, which then recruited the co-transcription factor HMGB2\nto the promoter region of the Lgr5 gene. This promoted transcription of the Lgr5 gene,\nwhich was crucial for stem cell self-renewal. The authors demonstrated that a new\nregulatory network involving Snora61-RBMX-HMGB2-Lgr5 contributes to ISC\nmaintenance. Overall, this study provides important insights into ISC self-renewal.\nHowever, several unresolved issues remain that need to be clarified.\n1. Why was analysis using snoRNA microarrays necessary? The authors should clarify\ntheir motivation. Furthermore, the expression of Snora61 in IEC is only twice that in ISC\n(Figure 1A). It is difficult to consider this significant. Further research should be conducted\nby comparing it in detail with other snoRNAs. The authors should include a table in the\nsupplementary materials showing the expression levels of other snoRNAs to make this\ninformation clear to readers.\nAnswer: This is a good question. Over the past decades, thousands of snoRNAs have\nbeen identified in the genome, yet the targets, mechanisms, and functions of most\nsnoRNAs remain unresolved. We compared snoRNA microarray data from intestinal stem\ncells and differentiated intestinal epithelial cells to identify snoRNAs highly enriched in\nintestinal stem cells, with the goal of screening for functionally important regulators of\nstemness. The expression levels in the microarray served as an initial screening criterion.\nWe designed shRNAs targeting the top 10 enriched snoRNAs and validated their\nknockdown efficiency and impact on organoid formation (new Fig. 1c and Supplementary\nFig. 1a, b). Since Snora61 knockdown exhibited the most significant downregulated effect\non organoid formation, we thus selected it for further investigation in this study. The full\nmicroarray dataset has been deposited in a public database for reader access.\n2. The Snora61 genes in humans and mice, designated NR_002987.1 and NR_034046.1,\nrespectively, exhibit significant sequence differences. The authors should clarify whether\nthey knocked out NR_034046.1 in mice or whether they knocked out another Snora61\nhomologous gene. This point should be explained in detail here, and further information\nshould be added to the text.\nAnswer: The sequence for mouse Snora61 provided in the NCBI database\n(NR_034046.1) is incomplete, containing only a partial segment, which may make\nmisunderstanding. For the generation of the Snora61 knockout mice, we referred to the\ncomplete sequence information available on the snoOPY database\n(http://snoopy.med.miyazakiu.ac.jp/snorna_db.cgi?mode=sno_info&id=Mus_musculus30\n0612) and the knockout strategy was illustrated in Supplementary Figure. 2a. The full\nsequence details for both the mouse Snora61 and human SNORA61 have been provided\nin the new Supplementary Figure 1c.\nThe grey areas indicate the overlapping regions:\nNR_034046.1: 5’-CCCTGAAGCTGGCCTCACAGCGATGCAGTCTGCTGTAGAGCAGC\nAGGCCAGACTCAGACATG-3’\nFull Snora61 sequence: 5’-ACCCTCCGGTTTCCCTTTCCCATCGGATCTTGACGCTG\nGTCTTGGTGGTCGTTAAGGAGGAAGAGCACCCTGAAGCTGGCCTCACAGCGATGCA\nGTCTGCTGTAGAGCAGCAGGCCAGACTCAGACATGC-3’\n3. As shown in Figure 3A, the ratio of Lgr5 expression levels between Snora61+/+ and\nSnora61/ mice is approximately twofold at most. Therefore, it is not necessary to proceed\nwith Lgr5 as the primary target. To thoroughly discuss this with readers, it is necessary to\npresent RNA-seq results confirming this finding in a table as supplementary material.\nAnswer: This is a good point. We reanalyzed the RNA-seq data in the new Figure 3.\nGO analysis revealed that genes downregulated upon Snora61 knockout were enriched in\npathways including gene transcription, cell proliferation/differentiation, and digestive tract\ndevelopment (new Figure. 3a). Further analysis of stemness-related genes indicated that\nLgr5 exhibited the most pronounced downregulation (new Figure. 3b). In addition, we\nadded RNA-seq of ISCs from Snora61+/+ and Snora61/ mice post irradiation. According\nto the RNA-seq results, a set of downstream target genes of LGR5 also showed various\ndegrees of downregulation, and these differences became more pronounced during the\nrepair process following irradiation-induced injury (new Figure. 3c, d). Consequently, the\ngene expression profile in Snora61/ ISCs showed significantly reduced enrichment in the\nWnt signaling pathway post irradiation (new Figure. 3e). Therefore, we focused on our\nmechanistic investigation of the transcriptional activation of Lgr5. The full RNA-seq dataset\nhas been deposited in a public database for reader access.\n4. The experimental methods are inadequately described. For this pull-down, the authors\nshould specify the amounts of biotin-labeled Snora61 and crypt cells used. In Figure 4A,\nwhich shows the experimental results, various different bands are observed in the\nexperimental and control groups. Many proteins other than RBMX must have bound. The\nauthors should present a table in the supplementary materials listing all identified proteins.\nAdditionally, they should provide detailed explanations regarding the criteria used to select\nRBMX from among these proteins.\nAnswer: We provided the relevant experimental details in the Methods section. RBMX\nwas not an arbitrary choice but a carefully selected Snora61-interacting protein identified\nthrough rigorous screening and validation. The entire pool of co-immunoprecipitated\nproteins was subjected to mass spectrometry analysis. We then conducted literature and\ndatabase research on the top-scoring candidates to assess their relevance to ISC self-\nrenewal and transcription regulation, which allowed us to preliminarily narrow down the list.\nFor proteins with potential roles, we further validated their interaction with Snora61 by\nimmunoblotting. In addition, we performed knockdown experiments to assess their impact\non ISC self-renewal using organoid formation assays. Through this stepwise screening\napproach, RBMX emerged as the sole candidate that both interacted with Snora61 and\nsignificantly affected organoid formation. Therefore, we selected RBMX as an interactor\nfor further investigation. The full mass spectrometry dataset has been deposited in a public\ndatabase for reader access.\n5. Figure 5A is very strange. HMGB2 is pulled down and visible as a band, but the band of\nRBMX, which should be co-precipitated, is not visible. Based on the principle of this\nexperiment, RBMX should precipitate more than HMGB2 because immunoprecipitation is\nperformed with an antibody against RBMX. The authors need to explain why HMGB2\nprecipitates in large amounts while RBMX does not.\nAnswer: In Figure. 5a, the arrow we drew indicates that HMGB2 was included within\nthe band at that band, rather than the entire band solely accounted for the HMGB2. In fact,\nthis band contained multiple proteins with similar molecular weights, which may lead to\nmisunderstanding (Attached Figure. 3a, b). The molecular weight of RBMX is 42.3 kDa,\nand its corresponding band is added in the gel image of Figure. 5a (Attached Figure. 3a).\nAdditionally, in the mass spectrometry analysis results, the most abundant protein\nidentified was also RBMX (Attached Figure. 3a, c). The full mass spectrometry dataset has\nbeen deposited in a public database for reader access.\nAttached Figures\nAttached Figure 1. Pull-down assay of mass spectrometry protein identification. (a)\nProtein identification corresponding to the 35 kDa band in Figure. 4a. (b) Protein\nidentification corresponding to the band above 55 kDa in Figure. 5a.\n✱✱✱\nAttached Figure 2. Snhg12 expression in mouse colon and SI ISCs. (a) RT-qPCR\nanalysis of Snhg12 expression in colon and SI ISCs. n=3 biological independent\nexperiments. *** P < 0.001. Data were analyzed by an unpaired two-side Student’s t-test\nand shown as means ± SD.\nAttached Figure 3. Co-IP assay of mass spectrometry protein identification. (a) Silver\nstaining image of co-immunoprecipitation (co-IP) assay of RBMX-binding proteins. (b)\nProtein identification corresponding to the 25 kDa band in Figure. 5a. (c) Protein\nidentification corresponding to the band above 40 kDa in Figure. 5a.\nPoint-by-point response to the reviewers\nReviewer #2:\nAlthough the authors have conducted substantial work, several of my key concerns\nremain unaddressed:\n1, As pointed out by Reviewer 1 and me, ChIRP-seq should have been performed.\nHowever, the authors did not carry out these experiments. Without the ChIRP-seq data,\nand given that the fold change of Lgr5 is very low in RNA-seq, the selected target Lgr5\nappears to be arbitrarily chosen rather than a bona fide target. Therefore, performing\nChIRP-seq is essential to convincingly demonstrate the direct targets of Snora61.\nAnswer: This is a good point. We provided the ChIRP-seq analysis of Snora61 in new\nFigure 3f. ChIRP-seq analysis revealed enrichment of Snora61 at the Lgr5 promoter region\n(new Fig. 3f). Our sequence alignment indicated complementary base-pairing between\nSnora61 and the enriched genomic segment (Fig. 3g). This binding and enrichment were\nfurther confirmed by ChIRP-qPCR and dot-blot assays, while mutation of the binding sites\nof Snora61 abolished the engagement (Fig. 3h, i). Based on these findings, and combined\nwith our analysis of the RNA-seq data, we conclude that Lgr5 is a bona fide target of\nSnora61. The raw ChIRP-seq data reported in this paper have been deposited in the GEO\ndatabase (GSE315692) that have been publicly accessible at\nhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE315692.\n2, The rescue experiments (comment 5 Figure 4) are unconvincing. After carefully\nexamining the shRNA sequences, I found that the shRNA targets the N-terminal domain of\nRBMX. In theory, any transcript of this gene would be susceptible to shRNA-mediated\ndegradation and therefore should not be expressed. Thus, the authors’ claim of restored\nexpression is mechanistically implausible and raises serious concerns about the validity of\nthese rescue experiments.\nAnswer: For the rescue experiments used in this study, synonymous mutations (i.e.,\ncodon substitutions) were introduced into the shRNA-targeted region during the design of\nthe overexpression plasmids. For Rbmx rescue, the sequence 5’-\nCGCCCAGGAAAGCTCTTTATT-3’ was replaced with 5’-CGGCCTGGTAAACTGTTCATT-\n3’, which ensured that the original shRNA did not interfere with the rescue effect. The\neffectiveness of the rescue experiments was validated by RT-qPCR in new Supplementary\nFig. 4i. We added the detailed methods in the Methods section.\n3, Some of the immunoblot data are missing, which is unacceptable for validating the\nauthors’ conclusions. LGR5 for Figure 3q; RBMX in Figure 4i from the same blot; HMGb2\nin Figure 5j from the same blot; Blot of HMBb2 and RBMX for Figure 5n.\nAnswer: We provided all requested Western blot data in the new Supplementary Fig.\n3l, Fig. 4i, Fig. 5j, and Supplementary Fig. 6f.\nReviewer #3:\nThe authors have addressed some of the concerns raised in my original review. But the\nfollowing important issues are not addressed. In particular, the authors argued that this\nstudy is focusing on intestine stem cells, but this cannot justify to examine other possible\neffects of Snora61 in the intestinal epithelial cells, which may contribute to stem cell self-\nrenewal and epithelial regeneration.\n1. Original Major Point 1:\n1) How to explain the differential expression pattern of Snora61 between the small and\nlarge intestinal epithelia? This question is not satisfactorily addressed. What causes the\ndifferential expression?\nAnswer: This is a very good point. Although small intestinal stem cells and colonic\nstem cells are both intestinal stem cells, they differ in their functional properties, cell fate\ndecisions, microenvironments (niches), as well as their proliferative demands (Gehart H.\n& Clevers H., Nat Rev Gastroenterol Hepatol, 2021). As such, small intestinal stem cells\nand colonic stem cells may have different gene profiles and intrinsic regulatory\nmechanisms. Regarding the differential expression levels of Snora61 in small intestinal\nstem cells versus colonic stem cells, we have two potential explanations as follows:\n(1)Different expression levels of the host gene between the small and large intestinal\nstem cells.RT-qPCR analysis revealed that Snhg12, the host gene encoding Snora61,\nwas much higher expression in the intestinal stem cells than in the colonic stem cells\n(Attached Fig. 1a). Through interrogating the ENCODE public database, we observed that\nabundant H3K4me3 enrichment at the transcription start site of Snhg12 in the small\nintestine (https://doi.org/doi:10.17989%2FENCSR000CCS), indicative of an open\nchromatin state in the small intestinal stem cells (Attached Fig. 1b). By contrast, ChIP\nexperiments showed that H3K4me3 levels at the corresponding promoter region were\nmarkedly lower in the colonic stem cells (Attached Fig. 1c), suggesting that Snhg12\nexpression was suppressed in these colonic stem cells. These results imply that\nexpression levels of host gene Snhg12 might account for the differential expression pattern\nof Snora61 between the small and large intestinal epithelia.\n(2)Potential difference of Snora61 generation between the small and large intestinal\nstem cells.\nAs for the snoRNA production, it is still elusive in the noncoding RNA field. It needs\nfurther investigation in the noncoding RNA field, which is beyond the scope of this study.\nAttached Figure 1. Snhg12 expression levels in mouse small intestinal stem cells versus colonic\nstem cells. (a) RT-qPCR analysis of Snhg12 expression in small intestinal stem cells (SI ISC) and colonic\nstem cells(Colon ISC). n=3 biological independent experiments. (b) H3K4me3 modification enrichment\npeaks at the Snhg12 promoter region (from ENCODE public database). (c) Enrichment analysis of\nH3K4me3 modification on Snhg12 promoter by ChIP-qPCR assay. n=3 biological independent\nexperiments. *** P < 0.001. Data were analyzed by an unpaired two-side Student’s t-test and shown as\nmeans ± SD.\n2) If the authors want to focus on Snora61 function in the regulation of small intestinal stem\ncells, then the title of this study should be changed to specifically indicate small intestine.\nAnswer: This is a good suggestion. We changed the title to “Small nucleolar RNA\nSnora61 drives self-renewal of small intestinal stem cells via initiation of Lgr5 transcription”.\n2. Original Major Point 5: As Snora61 regulates both self-renewal and cell proliferation, this\nfact should be clearly stated in the Abstract to avoid the misleading that Snora61 only\nregulates self-renewal.\nAnswer: We revised these descriptions in the Abstract section: Snora61 promotes the\nself-renewal of small intestinal stem cells, which in turn enhances the proliferation of their\ndifferentiated intestinal epithelial cells, contributing to the maintenance of intestinal\nhomeostasis.\n3. Original Major Point 7: Snora61 KO only causes 50% decrease of LGR5 expression.\nThe authors stated “we focused on the mechanism by which Snora61 influenced intestinal\nstem cell stemness via LGR5, Snora61 may also contribute to intestinal stem cell\nhomeostasis through additional pathways” in the rebuttal. Based on this statement, the\ninfluence of Snora61 on intestinal stem cell stemness via LGR5 is questionable. Also, they\nshould show what are the possible other additional pathways.\nAnswer: This is a good point. While analyzing the RNA-seq data, we noticed that\nSnora61 knockout altered the expression of several stemness-related genes (including\nLgr5, Msi1, Tert, Clca4a, Olfm4, Ascl2, Cdca7, Axin2, Smoc2, Lrig1, and Sox9) (Fig. 3b).\nAmong these genes, Lgr5 exhibited the most pronounced change. Furthermore, our\nChIRP-seq results demonstrated that Lgr5 is a bona fide target of Snora61 (Fig. 3f) as\nanswered in the Reviewer#2. Thus, we selected Lgr5 as a direct target of Snora61 in the\nregulation of intestinal stem cell stemness.\nIndeed, Snora61 could also affect intestinal stem cell homeostasis through additional\npathways. Our GO analysis indicated that, in addition to stemness regulation, genes\ndownregulated upon Snora61 knockout were also involved in other pathways such as\nnutrient absorption, energy metabolism and protein phosphorylation (Fig. 3a). We\naddressed this issue in the Discussion section.\n4. Original Major Point 13: The possible effect of Snora61 on other genes/pathways beside\nLGR5 should be discussed in the Discussion.\nAnswer: We addressed this issue in the Discussion section.\nReviewer #4:\nThe reviewer expresses concern over the authors' failure to provide straightforward\nanswers. The reviewer's responses to the aforementioned inquiries are detailed below.\n1. The authors have not addressed some of this reviewer's concerns. Again, the expression\nlevel of Snora61 in IEC is only twice that in ISC (Figure 1A). Furthermore, while this\nexperiment appears to have been performed three times, error bars are not shown in\nFigure 1A. No data indicating that this twofold difference is statistically significant was found.\nThe authors need to demonstrate that this twofold difference is significant or write this\npaper excluding this data. This microarray dataset was not found in public databases.\nAnswer: We conducted an initial screening for snoRNAs highly expressed in intestinal\nstem cells using snoRNA microarray analysis. Since we only used one pair of samples,\nthere were no error bars in Figure 1a. The significant high expression of Snora61 in\nintestinal stem cells was validated through RT-qPCR as shown in new Figure 1b. We\nvalidated that Snora61 was about 5 times higher in LGR5+ ISCs versus LGR5- ISCs. The\nraw sequence data reported in this paper have been deposited in the Genome Sequence\nArchive in National Genomics Data Center, China National Center for Bioinformation /\nBeijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA033396) that have\nbeen publicly accessible athttps://ngdc.cncb.ac.cn/gsa.\n2. The reviewer understands the authors' explanation regarding the differences between\nhuman and mouse Snora61. However, the reviewer has found that the procedure for the\nmouse Snora61 knockout is not described in the Methods section. According to Figure S2a,\nit appears the authors created a partial deletion mutant of Snora61. The method used to\ncreate this should be described. Additionally, the authors should add information to Figure\nS2a regarding the type, location, and size of the drug resistance gene used for the\nscreening.\nAnswer: The Snora61/ mice were generated by the gene editing facility of our\nInstitute of Biophysics using the CRISPR/Cas9 technology. The specific procedure is as\nfollows: the sgRNAs targeting Snora61 were designed by online CRISPR design tool\n(https://crispor.gi.ucsc.edu/). The sgRNA sequences are listed in Supplementary Table 1.\nApproximately 250 zygotes in the C57BL/6 background were injected with corresponding\nsingle guide RNA (sgRNA) donors and subsequently transferred to the uterus of pseudo-\npregnant ICR females, from which viable founder mice were obtained. Knockout efficiency\nin the offspring mice was verified by PCR and next-generation sequencing, rather than\nthrough drug-resistance gene screening. The genomic sequence alignment results of the\nSnora61 locus in the knockout mice showed that, the vast majority of the Snora61\nsequence had been successfully deleted (only 22 bp remained, which was insufficient to\nkeep a functional role) (Attached Fig. 2). We provided detailed methodology for generating\nthe Snora61 knockout mice in the Methods section.\nAttached Figure 2. Genomic sequence alignment of the Snora61 locus in Snora61/ mice.\nSequence alignment was performed using the SnapGene software.\n3. The authors did not address the reviewer's inquiries. There is no mention of the ratio of\nLgr5 expression levels/sequence counts between Snora61+/+ and Snora61/ mice in the\nRNA-seq data. Therefore, there is no compelling necessity to proceed with subsequent\nanalyses treating Lgr5 as the primary target, and the approach taken in this paper is not\nsupported. No publicly available database containing the full RNA-seq dataset was found.\nAnswer: We presented the differential expression analysis of stemness-related genes\n(including Lgr5) derived from the RNA-seq data in Figure 3b. The log fold change of Lgr5\n2\nexpression in Snora61/ and Snora61+/+ ISCs was approximately -0.75, which\ncorresponded to a ratio of about 0.59. Among the stemness-related genes, Lgr5 exhibited\nthe most pronounced change. In addition, Lgr5and its downstream target genes showed\nvarious degrees of downregulation, which was particularly evident during post-irradiation\nrepair (Fig. 3c, d). Consequently, the gene expression profile in Snora61/ ISCs showed\nsignificantly reduced enrichment in the Wnt signaling pathway post irradiation (Fig. 3e).\nFurthermore, we added ChIRP-seq analysis of Snora61 in new Figure 3f. ChIRP-seq\nanalysis and related assay results provided direct evidence linking Snora61 to the\ntranscriptional regulation of Lgr5: ChIRP-seq analysis revealed enrichment of Snora61 at\nthe Lgr5 promoter region (new Fig. 3f). Sequence alignment indicated complementary\nbase-pairing between Snora61 and the enriched genomic segment (Fig. 3g). This binding\nand enrichment were further confirmed by ChIRP-qPCR and dot-blot assays, while\nmutation of the binding sites of Snora61 abolished the engagement (Fig. 3h, i). Based on\nthese findings, we thus selected Lgr5 as the primary focus for our mechanistic investigation.\nThe raw RNA-seq data reported in this paper have been deposited in the Genome\nSequence Archive in National Genomics Data Center, China National Center for\nBioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA:\nCRA026331, CRA033390, CRA033391) that have been publicly accessible\nathttps://ngdc.cncb.ac.cn/gsa. The raw ChIRP-seq data reported in this paper have been\ndeposited in the GEO database (GSE315692) that have been publicly accessible at\nhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE315692.\n4. The authors must provide data demonstrating that \"RBMX was a carefully selected,\nrigorously screened and validated Snora61-interacting protein.\" Currently, the objectivity of\nthis process is not assured. The supplementary materials should list all interacting proteins\nand explain how RBMX was identified as a candidate. The mass spectrometry dataset\ncould not be found in public databases.\nAnswer: Following the RNA pull-down assay, we cut the two most prominent\ndifferential bands from the silver-stained gel — above 40 kDa and below 35 kDa in the\n“Sense” lane (as shown in Figure 4a) — for mass spectrometry analysis. In order to identify\nSnora61-interacting protein candidates, we first filtered for proteins unmatching the\nexpected molecular weight ranges of the excised bands. The remaining candidate proteins\nwere listed in the Supplementary Table 6. We then excluded five proteins with low\nconfidence scores (LYAR, PP-1, RFC2, MARE2, and CENPV). Given that Snora61 is\nlocated in the nucleus, we conducted further screening based on the location of the protein\nwithin the cell. Through database and literature review, we noticed that K1C19 and TPM1\nare structural proteins localized in the cytoplasm; RS3A is a ribosomal structural protein\nwhose mature form is predominantly found in cytoplasmic ribosomes, therefore, they were\nexcluded from further consideration. Both RBMX and HSPA8 have been reported to\nlocalize to the nucleus, with RBMX being a well-characterized nuclear protein and HSPA8\nprimarily cytoplasmic but also present at low levels in the nucleus. To identify candidate\nproteins capable of regulating Lgr5 transcription, we designed shRNAs targeting Hspa8\nand Rbmx and examined their knockdown effects on Lgr5 expression. The results showed\nthat Hspa8 knockdown did not affect Lgr5 expression, whereas Rbmx knockdown\nsignificantly suppressed Lgr5 expression (Supplementary Fig. 4a). In addition, we further\nvalidated the interaction between Snora61 and RBMX in Figure 4b-d. Therefore, we\nultimately selected RBMX as the Snora61-interacting protein in this study.\nThe mass spectrometry data have been deposited to the ProteomeXchange\nConsortium (https://proteomecentral.proteomexchange.org) via the iProX partner\nrepository with the dataset identifierPXD070722, which has been publicly accessible.\n5. Figure 5A shows only a single band at 25kDa. This pattern differs from the typical\noverlap of multiple protein bands with similar molecular weights. Therefore, HMGB2 is\nconsidered the major component of this band. Based on this assumption, RBMX should\nprecipitate more than HMGB2 because the antibody binds to both RBMX with and without\nHMGB2. However, the intensity of the RBMX and HMGB2 bands in Figure 5A does not\nmatch this expectation. This contradicts the authors' explanation. If the HMGB2 band were\ncontaminated with other proteins --- background due to non-specific adsorption --- of\ncomparable quantity, such contamination would need to be visible in the control lane at the\n25 kDa position. However, no such band is visible in the control, contradicting the\nauthors' reply. Therefore, the data in Figure 5A is flawed, and the authors should exclude\nthis data and reconstruct their paper. The mass spectrometry dataset could not be found\nin public databases.\nAnswer: This is a good point. We optimized the experimental conditions and repeated\nthe co-immunoprecipitation (co-IP) assay as shown in new Figure 5a.\nThe mass spectrometry proteomics data have been deposited to the\nProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the\niProX partner repository with the dataset identifierPXD070722, which has been publicly\naccessible.\nPoint-by-point response to the reviewers\nReviewer #4:\nAlthough the authors have conducted substantial work, several of my key concerns remain\nunaddressed:\n1. The subsequent RT-qPCR that the authors cite as a rebuttal does not validate the\naccuracy of the microarray assay. The reviewer suggests removing Figure 1A. Figure 1A\nshould serve as the starting point for all subsequent studies. Many researchers cannot use\na single experiment showing a twofold difference in gene expression as the basis for their\nresearch. Multiple replicates would likely be required even for submission to other\nspecialized journals.\nAnswer: Thank you for your suggestion. The microarray assay results served as a\npreliminaryscreening step to identify snoRNAs enriched in intestinal stem cells. Our\nsubsequent RT qPCR validation and organoid formation assays were based on the results\nof this screening step, therefore removing these data may not be appropriate for the logic\n-\nof this study.\n2. The reviewer agreed with the authors' comments.\n3. The reviewer suggests deleting Figure 3B. As the authors commented, the ratio of Lgr5\nexpression levels between Snora61+/+ and Snora61-/- mice is 0.59-fold. Many\nresearchers would not consider a single experiment demonstrating a 0.59-fold difference\nin expression levels as sufficient justification for initiating a series of follow-up experiments.\nFigure 3B currently presents unconvincing data.\nAnswer: Figure 3b presents the expression trends of stemness related genes in\nintestinal stem cells, serving as preliminary guidance for identifying candidate target genes.\n-\nAfter focusing our investigation on Lgr5, we further assessed its expression levels under\nboth homeostatic conditions and post irradiation. These results, along with statistical\nsignificance tests, are presented in Figures 3c, 3d, and Supplementary Figure 3g to\ndemonstrate the significant changes. If you think it would be better to move the original\nFigure 3b to the supplementary materials, we will follow your suggestion.\n4. The authors' rebuttal is contradictory. Figure 4A shows various Sense-specific\nprecipitated proteins appearing in the 50–60 kDa range and the range below 30 kDa.\nHowever, these were not included in Supplementary Table 6. This is despite the authors\nstating that the predicted molecular weight range of the excised bands was below 35 kDa\nand above 40 kDa. The reviewers could not understand why RBMX was solely presented\nas the putative candidate.\nAnswer: I sincerely apologize for not clearly explaining our experimental methodology\nin the previous response letter and the Methods section. For mass spectrometry analysis\nof this silver staining image, we only cut the two most prominent differential bands around\n42 kDa and 30 kDa to conduct mass spectrometry. The remaining candidate proteins were\nlisted in the Supplementary Table 6. As answered at the last response letter, we selected\nand validated the RBMX as the Snora61-interacting protein in this study. Since the Sense-\nspecific precipitated proteins appearing in the 50–60 kDa range and the range far below\n30 kDa were not differentially remarked, we just ignored these bands for mass\nspectrometry. The Supplementary Table 6 includes all candidate proteins of the two most\nprominent differential bands around 42 kDa and 30 kDa. We addressed this issue in the\nMethods section.\nAttached figure 1: Cut strategy of silver staining image of pull-down assay for Snora61-binding\nproteins. Two bands cut for mass spectrometry analysis are marked with red boxes.\n5. Since the issue remains unresolved, the reviewer will provide comments similar to the\nprevious ones. Figure 5A shows only a single band at 25k Da. This differs from the typical\noverlap of multiple protein bands with similar molecular weights. Therefore, HMGB2 is\nconsidered the major component of this band. Based on this assumption, RBMX should\nprecipitate more than HMGB2 because the antibody binds to both RBMX with and without\nHMGB2. However, the intensity of the RBMX and HMGB2 bands in Figure 5A does not\nmatch this expectation. Therefore, the data in Figure 5A is flawed, and the authors should\nexclude this data and reconstruct their paper. Alternatively, they should seek assistance\nfrom researchers in this field, as they appear to have limited experience with proteomics\nresearch.\nAnswer: These is a very good point. The mass spectrometry and sequence analysis\nwere conducted by our institutional experts of the Institute of Biophysics, who served over\n200 labs nationwide and published over 100 high profile papers each year. In our\nexperimental methodology, we performed co-IP assay using a 12% polyacrylamide gel for\nprotein separation. In the 12% polyacrylamide gel electrophoresis, proteins with close\nmolecular weights might migrate as an unresolved single band. In fact, the most prominent\ndifferential band around 25 kDa band in the Figure 5a was contained several candidate\nproteins with molecular weights close to HMGB2 (around 0.5 kDa size difference) (new\nSupplementary Table 7). We aimed to identify proteins with potential regulatory effects on\nLgr5 transcription through co-IP assay. After evaluating the subcellular localization and\ntranscriptional activity of the candidate proteins, we ultimately identified and confirmed\nHMGB2 as the binding protein of RBMX in the nucleus of ISCs.\nIn fact, anti-RBMX antibody precipitated more RBMX than HMGB2 in this assay. We\nidentified RBMX peptide by mass spectrometry in the band at the 42 kDa position with a\nhigh coverage score—23.05, while the score of HMGB2 detected from the differential band\nwas 7.58. The score of RBMX was much higher than that of HMGB2. In addition, the\ndifferential band around 25 kDa contained several proteins with close molecular weights\nincluding RL13, RAN, HMGB2, Pentraxin family member and Keratin 80, thus the band of\nHMGB2 in this image was denser than that of RBMX. We provided relevant mass\nspectrometry data in the new Supplementary Table 7 and detailed supplementary\ndescriptions of the co-IP assay in both the main text and the Methods section.\nSupplementary Table 7: Coverage scores of candidate binding proteins obtained\nfrom co-IP assays in Figure 5a\nProtein name MW [kDa] Score Accession\nRBMX 42.3 23.05 Q9WV02\nRL13 24.6 10.56 Q5RKP3\nRAN 24.4 9.17 P62827\nHMGB2 24.2 7.58 P30681\nPentraxin family member 24.5 4.23 D3YYJ7\nKeratin 80 24.7 2.69 Q3TBQ3"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I have carefully evaluated the authors’ revisions and find that they have adequately addressed all of my previous comments.\nThe manuscript has been improved and is now suitable for publication.", "reviewer_2": "Although the authors have conducted substantial work, several of my key concerns remain unaddressed:\n1, As pointed out by Reviewer 1 and me, ChIRP-seq should have been performed. However, the authors did not carry out these experiments. Without the ChIRP-seq data, and given that the fold change of Lgr5 is very low in RNA-seq, the selected target Lgr5 appears to be arbitrarily chosen rather than a bona fide target. Therefore, performing ChIP-seq is essential to convincingly demonstrate the direct targets of Snora61.\n2, The rescue experiments (comment 5 Figure 4) are unconvincing. After carefully examining the shRNA sequences, I found that the shRNA targets the N-terminal domain of RBMX. In theory, any transcript of this gene would be susceptible to shRNA-mediated degradation and therefore should not be expressed. Thus, the authors’ claim of restored expression is mechanistically implausible and raises serious concerns about the validity of these rescue experiments.\n3, Some of the immunoblot data are missing, which is unacceptable for validating the authors’ conclusions. LGR5 for Figure 3q; RBMX in Figure 4i from the same blot; HMGb2 in Figure 5j from the same blot; Blot of HMBb2 and RBMX for Figure 5n.", "reviewer_3": "The authors have addressed some of the concerns raised in my original review. But the following important issues are not addressed. In particular, the authors argued that this study is focusing on intestine stem cells, but this cannot justify to examine other possible effects of Snora61 in the intestinal epithelial cells, which may contribute to stem cell self-renewal and epithelial regeneration.\n1. Original Major Point 1: 1) How to explain the differential expression pattern of Snora61 between the small and large intestinal epithelia?\nThis question is not satisfactorily addressed. What causes the differential expression?\n2) If the authors want to focus on Snora61 function in the regulation of small intestinal stem cells, then the title of this study should be changed to specifically indicate small intestine.\n2. Original Major Point 5: As Snora61 regulates both self-renewal and cell proliferation, this fact should be clearly stated in the Abstract to avoid the misleading that Snora61 only regulates self-renewal.\n3. Original Major Point 7: Snora61 KO only causes 50% decrease of LGR5 expression. The authors stated “we focused on the mechanism by which Snora61 influenced intestinal stem cell stemness via LGR5, Snora61 may also contribute to intestinal stem cell homeostasis through additional pathways” in the rebuttal. Based on this statement, the influence of Snora61 on intestinal stem cell stemness via LGR5 is questionable. Also, they should show what are the possible other additional pathways.\n4. Original Major Point 13: The possible effect of Snora61 on other genes/pathways beside Lgr5 should be discussed in the Discussion.", "reviewer_4": "The reviewer expresses concern over the authors' failure to provide straightforward answers. The reviewer's responses to the aforementioned inquiries are detailed below.\n1. The authors have not addressed some of this reviewer's concerns. Again, the expression level of Snora61 in IEC is only twice that in ISC (Figure 1A). Furthermore, while this experiment appears to have been performed three times, error bars are not shown in Figure 1A. No data indicating that this twofold difference is statistically significant was found. The authors need to demonstrate that this twofold difference is significant or write this paper excluding this data. This microarray dataset was not found in public databases.\n2. The reviewer understands the authors' explanation regarding the differences between human and mouse snoR61.\nHowever, the reviewer has found that the procedure for the mouse snoR61 knockout is not described in the Methods section. According to Figure S2a, it appears the authors created a partial deletion mutant of snoR61. The method used to create this should be described. Additionally, the authors should add information to Figure S2a regarding the type, location, and size of the drug resistance gene used for the screening.\n3. The authors did not address the reviewer's inquiries. There is no mention of the ratio of Lgr5 expression levels/sequence counts between Snora61+/+ and Snora61-/- mice in the RNA-seq data. Therefore, there is no compelling necessity to proceed with subsequent analyses treating Lgr5 as the primary target, and the approach taken in this paper is not supported. No publicly available database containing the full RNA-seq dataset was found.\n4. The authors must provide data demonstrating that \"RBMX was a carefully selected, rigorously screened and validated Snora61-interacting protein.\" Currently, the objectivity of this process is not assured. The supplementary materials should list all interacting proteins and explain how RBMX was identified as a candidate. The mass spectrometry dataset could not be found in public databases.\n5. Figure 5A shows only a single band at 25k Da. This pattern differs from the typical overlap of multiple protein bands with similar molecular weights. Therefore, HMGB2 is considered the major component of this band. Based on this assumption, RBMX should precipitate more than HMGB2. However, the intensity of the RBMX and HMGB2 bands in Figure 5A does not match this expectation. This contradicts the authors' explanation. If the HMGB2 band were contaminated with other proteins --- background due to non-specific adsorption --- of comparable quantity, such contamination would need to be visible in the control lane at the 25 kDa positoin. However, no such band is visible in the control, contradicting the authors' reply. Therefore, the data in Figure 5A is flawed, and the authors should exclude this data and reconstruct their paper. The mass spectrometry dataset could not be found in public databases."}, "author_response": ""}, {"round": 3, "reviewer_comments": {"reviewer_2": "I would like to thank the authors for their work and their answers to the concerns.", "reviewer_3": "no more questions", "reviewer_4": "Although the authors have conducted substantial work, several of my key concerns remain unaddressed:\n1. The subsequent RT-qPCR that the authors cite as a rebuttal does not validate the accuracy of the microarray assay. The reviewer suggests removing Figure 1A. Figure 1A should serve as the starting point for all subsequent studies. Many researchers cannot use a single experiment showing a twofold difference in gene expression as the basis for their research. Multiple replicates would likely be required even for submission to other specialized journals.\n2. The reviewer agreed with the authors' comments.\n3. The reviewer suggests deleting Figure 3B. As the authors commented, the ratio of Lgr5 expression levels between Snora61+/+ and Snora61-/- mice is 0.59-fold. Many researchers would not consider a single experiment demonstrating a 0.59-fold difference in expression levels as sufficient justification for initiating a series of follow-up experiments. Figure 3B currently presents unconvincing data.\n4. The authors' rebuttal is contradictory. Figure 4A shows various Sense-specific precipitated proteins appearing in the 50–60 kDa range and the range below 30 kDa. However, these were not included in Supplementary Table 6. This is despite the authors stating that the predicted molecular weight range of the excised bands was below 35 kDa and above 40 kDa. The reviewers could not understand why RBMX was solely presented as the putative candidate.\n5. Since the issue remains unresolved, the reviewer will provide comments similar to the previous ones. Figure 5A shows only a single band at 25k Da. This differs from the typical overlap of multiple protein bands with similar molecular weights. Therefore, HMGB2 is considered the major component of this band. Based on this assumption, RBMX should precipitate more than HMGB2 because the antibody binds to both RBMX with and without HMGB2. However, the intensity of the RBMX and HMGB2 bands in Figure 5A does not match this expectation. Therefore, the data in Figure 5A is flawed, and the authors should exclude this data and reconstruct their paper. Alternatively, they should seek assistance from researchers in this field, as they appear to have limited experience with proteomics research."}, "author_response": ""}]

Decision letter