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LSM7 Coordinates scaRNA-Mediated snRNA Modification to Ensure Spliceosome Fidelity and Spermatogonial Stem Cell Differentiation [RIP-Seq]

ABSTRACT: Spermatogenesis requires precise spatial and temporal regulation of RNA splicing, orchestrated by both the major (U1, U2, and U4/U6/U5) and minor (U11, U12, and U4atac/U6atac/U5) spliceosomal small nuclear ribonucleoproteins (snRNPs), yet their function during germ cell development remain poorly defined. Here, we elucidate the essential function of LSM7, a component of the LSM2-8 complex, in snRNP biogenesis and the process of spermatogenesis. Our results demonstrate that LSM7 governs the stability of U6 small nuclear RNA (snRNA) within Cajal bodies and directly interacts with scaRNA2, scaRNA13, and scaRNA17 to modulate 2′-O-methylation (2′-O-Me) and pseudouridylation (Ψ) modifications of U2 and U12 snRNAs. Loss of LSM7 results in defective nuclear localization and reduced stability of U6, U2, U5, and U12 snRNAs, destabilization of their corresponding snRNP complexes, and aberrant splicing events, notably affecting mutually exclusive exons (MXE) and exon skipping (SE). These splicing defects lead to translational dysregulation, ultimately impairing the differentiation of spermatogonial stem cells (SSCs) into spermatogonia. Our findings identify LSM7 as a pivotal regulator linking scaRNA-guided snRNA modifications, spliceosomal fidelity, and the determination of spermatogonial stem cell fate, offering important mechanistic insights into the post-transcriptional regulation of male germ cell development.

ORGANISM(S): Mus musculus

PROVIDER: GSE304555 | GEO | 2026/05/20

REPOSITORIES: GEO

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LSM7 Coordinates scaRNA-Mediated snRNA Modification to Ensure Spliceosome Fidelity and Spermatogonial Stem Cell Differentiation

Project description:Spermatogenesis requires precise spatial and temporal regulation of RNA splicing, orchestrated by both the major (U1, U2, and U4/U6/U5) and minor (U11, U12, and U4atac/U6atac/U5) spliceosomal small nuclear ribonucleoproteins (snRNPs), yet their function during germ cell development remain poorly defined. Here, we elucidate the essential function of LSM7, a component of the LSM2-8 complex, in snRNP biogenesis and the process of spermatogenesis. Our results demonstrate that LSM7 governs the stability of U6 small nuclear RNA (snRNA) within Cajal bodies and directly interacts with scaRNA2, scaRNA13, and scaRNA17 to modulate 2′-O-methylation (2′-O-Me) and pseudouridylation (Ψ) modifications of U2 and U12 snRNAs. Loss of LSM7 results in defective nuclear localization and reduced stability of U6, U2, U5, and U12 snRNAs, destabilization of their corresponding snRNP complexes, and aberrant splicing events, notably affecting mutually exclusive exons (MXE) and exon skipping (SE). These splicing defects lead to translational dysregulation, ultimately impairing the differentiation of spermatogonial stem cells (SSCs) into spermatogonia. Our findings identify LSM7 as a pivotal regulator linking scaRNA-guided snRNA modifications, spliceosomal fidelity, and the determination of spermatogonial stem cell fate, offering important mechanistic insights into the post-transcriptional regulation of male germ cell development.

2026-05-20 | GSE304556 | GEO

Nopp140-chaperoned 2’-O-methylation of small nuclear RNAs in Cajal bodies ensures splicing fidelity

Project description:Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB) specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at some 80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2’-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.

2021-07-01 | GSE173171 | GEO

Transcriptome changes in splicing inhibited cells

Project description:In eukaryotes, U1 small nuclear ribonucleoprotein (snRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 snRNPs; however, its abundance in human far exceeds that of the other snRNPs. Here we used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 snRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (<5 kilobases) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA antisense morpholino oligonucleotide or the U2-snRNP-inactivating drug spliceostatin A unless U1 antisense morpholino oligonucleotide was also included. We further show that U1 snRNA–pre-mRNA base pairing was required to suppress premature cleavage and polyadenylation from nearby cryptic polyadenylation signals located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.

2010-09-29 | GSE24179 | GEO

Suppression of prp28-1 splicing defect by prp8-tes allele

Project description:DEAD-box proteins, a family of RNA-dependent ATPases, promote the numerous conformational rearrangements required for spliceosome assembly, activation, and disassembly. Previous work showed that a cold-sensitive substitution in DEAD-box protein Prp28 prevents the switch from U1 to U6 snRNA pairing with the 5’ splice site. Little is known about how Prp28 is regulated, although U5 snRNP protein Prp8 is a potential coordinator of Prp28 and other spliceosomal ATPases. We conducted a targeted selection in Prp8 for cold-insensitive suppressors of prp28-1, then used splicing specific microarrays to assess suppression of the prp28-1 splicing defect. Splicing specific microarrays were used to assess suppression of the prp28-1 splicing defect by a prp8 allele (prp8-tes) that suppresses prp28-1 cold-sensitivity

2014-01-01 | E-GEOD-42754 | biostudies-arrayexpress

Biallelic mutations in the 3’ exonuclease TOE1 cause Pontocerebellar Hypoplasia Type 7 and result in snRNA processing defects

Project description:Processing of RNA polymerase II-transcribed spliceosomal small nuclear RNAs (snRNAs) initiates with cleavage by the Integrator complex, but the factors that subsequently remove 3’ end extensions from metazoan snRNAs have remained unknown. We studied human families with a unique recessive syndromic constellation of pontocerebellar atrophy with ambiguous genitalia, uncovering biallelic inactivating mutations in TOE1, which encodes a conserved unconventional deadenylase. TOE1 demonstrated tight association with snRNA-protein (snRNP) complexes with specificity for incompletely processed pre-snRNAs containing 3’ end extensions that often included post-transcriptionally added tails. Human cells deficient for TOE1 catalytic activity showed accumulation of 3’-end extended U1, U2, U4 and U5 pre-snRNAs, and TOE1 immuno-isolated from human cells was capable of processing 3’-end extended snRNPs in vitro. The missense mutations identified in patients impaired TOE1 stability and snRNA processing in patient cells, which was associated with defects in splicing. Our findings reveal the cause of a unique brain malformation and uncover a long-sought 3’ exonuclease required for snRNA processing.

2016-12-31 | GSE71536 | GEO

RiboMeth-Seq analysis of purified U6 and U2 snRNAs

Project description:The La-related protein LARP7 has been mainly described as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, which negatively regulates RNA polymerase II by sequestering the positive transcription elongation factor b (P-TEFb). In our studies, we discovered a novel, 7SK snRNP-independent function of LARP7. We show that LARP7 interacts with the U6 spliceosomal RNA as well as with the small nucleolar RNAs (snoRNAs) directing the 2'-O-methylations of U6. To investigate the relevance of this interaction, U6 or U2 snRNAs were purified from total RNA by pulldown of biotinylated antisense oligonucleotides and the occurence of 2’-O-methylations was investigated by RiboMeth-seq analysis. A comparison between U6 and U2 snRNA isolated from HEK293 wildtype or LARP7 knockout cell lines revealed that 2’-O-methylations of the U6 snRNA are specifically lost in the absence of LARP7. Alazami syndrome is a form of primary dwarfism associated with mutations in the LARP7 gene. RiboMeth-seq analyses performed with RNA isolated from blood samples of two Alazami patients or healthy parents as well as from B-lymphoblastoid cell lines (B-LCLs) derived from an Alazami patient and from a healthy parent confirmed the impact of mutant LARP7 protein variants on the 2’-O-methylation profile of the U6 snRNA.

2020-02-03 | GSE126593 | GEO

The structure and target identification of the tandem RRM of yeast Npl3 bound to RNA reveal a cooperative sequence-specific recognition and an RNA chaperone role in splicing

Project description:The conserved SR-like protein Npl3 promotes the splicing of diverse pre-mRNAs. However, the RNA sequence(s) recognized by the RNA Recognition Motifs (RRM1 & RRM2) of Npl3 during the splicing reaction remain elusive. Here, we developed a split-iCRAC approach in vivo to determine the consensus sequence bound to each RRM in yeast. High-resolution NMR structures show that RRM2 recognizes a 5´-GNGG-3´ motif leading to an unusual mille-feuille topology. In addition, our data indicate a non-specific interaction of the RS domain with RNA. These structures reveal how RRM1 preferentially interacts with a CC-dinucleotide upstream of this motif, and how the inter-RRM linker and the region C-terminal to RRM2 contributes to cooperative RNA-binding. Structure-guided studies show that Npl3 genetically interacts with U2 snRNP specific factors and we provide evidence that Npl3 melts U2 snRNA stem-loop I, a prerequisite for U2/U6 duplex formation within the catalytic centre of the Bact spliceosomal complex. Our findings provide a mechanistic role for Npl3 during spliceosome active site formation.

2022-12-01 | E-MTAB-11736 | biostudies-arrayexpress

SILAC APMS anlysis of TSSC4 interactome

Project description:U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo an intricate biogenesis that ensures that only a fully mature particle assembles into a splicing competent U4/U6•U5 tri-snRNP and enters the splicing reaction. During splicing, U5 snRNP is substantially rearranged and leaves as a U5/PRPF19 post-splicing particle, which requires re-generation before a next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a new component of U5 snRNP that promotes tri-snRNP formation. We provide evidence that TSSC4 associates with U5 snRNP chaperones, U5 snRNP and the U5/PRPF19 particle. Specifically, TSSC4 interacts with U5-specific proteins PRPF8, EFTUD2 and SNRNP200. We also identified TSSC4 domains critical for the interaction with U5 snRNP and the PRPF19 complex, as well as for TSSC4 function in tri-snRNP assembly. TSSC4 emerges as a specific chaperone that acts in U5 snRNP de novo biogenesis as well as post-splicing recycling.

2021-06-11 | PXD024929 | Pride

Suppression of prp28-1 splicing defect by prp8-tes allele

2014-01-01 | GSE42754 | GEO

The Role of Extracellular Spliceosomal snRNAs in Communication of Ovarian Cancer Cells

Project description:Exogenous signals from drug-stressed cancer cells accelerate the proliferation of neighboring tumor cells and promote them to acquire a more aggressive phenotype. We have recently found an exciting phenomenon: drug-stressed cancer cells secrete various components of the spliceosome: proteins and a number of snRNAs. We have observed this phenomenon both in vitro (culture media from cancer cell lines [Pavluykov M. et al.//Cancer Cell, 2018]) and in vivo (ovarian cancer ascites after chemotherapy [Shender V. et al//Mol. Сell. Proteomics, 2014]). The aim of this study was to elucidate the role of secreted spliceosomal snRNAs in intercellular communication. We constructed synthetic U12 and U6atac snRNAs close to the natural structure, including some non-canonical nucleotides imitating post-transcriptional RNA modification. Proteomic analysis of SKOV3 cells transfected with U6atac or U12 snRNA analogs has shown that both exogenous snRNAs lead to an increase of abundance of proteins related to cell cycle regulation and M phase. It has been recently shown that a number of spliceosomal proteins affect regulation of the cell cycle, in particular the M phase (Maslon et al., 2014). Here we demonstrated that not only spliceosomal proteins but also spliceosomal snRNAs (U12 and U6atac) affect activation of mitotic cell cycle genes. This study reveals previously unknown signaling molecules in the microenvironment of ovarian cancer that have potential clinical significance.

2024-05-29 | PXD027950 | Pride

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