FIGURE 6 - uploaded by Claudia Schneider
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The 12S U11 snRNP contains a subset of U11/U12 proteins. ( A ) Proteins from purified 18S U11/U12 snRNPs (lanes 1 , 2 , 4 , 6 , 8 , 10 , 12 , 14 ) or snRNPs enriched in 12S U11 snRNPs (lanes 3 , 5 , 7 , 9 , 11 , 13 , 15 ) were stained with Ponceau S (lane 1 ) or antibodies against the protein indicated at the top . The positions of the U11/U12 proteins are indicated on the left . ( B ) Immunoprecipitations were performed with immunoaffinity-purified, glycerol gradient- fractionated snRNPs from the 18S ( top ) or 12S ( bottom ) region of the gradient and immunoaffinity-purified antibodies against the 35K (lane 3 ), 25K (lane 4 ), 20K (lane 5 ), 59K (lane 6 ) protein, or PAS beads alone (lane 2 ). (Lane 1 ) 1 ⁄ 40 or 1 ⁄ 25 of the input 18S or 12S gradient
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U11 and U12 snRNPs bind U12-type pre-mRNAs as a preformed di-snRNP complex, simultaneously recognizing the 5' splice site and branchpoint sequence. Thus, within the U12-type prespliceosome, U11/U12 components form a molecular bridge connecting both ends of the intron. We have affinity purified human 18S U11/U12 and 12S U11 snRNPs, and identified th...
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... characterize the novel U11/U12 proteins in more detail, we raised antibodies against each of them. Antibodies against the 65K, 59K, 48K, 35K, 31K, 25K, or 20K proteins reacted with the expected U11/U12 protein on immuno- blots containing U11/U12 proteins ( Fig. 6A, even lanes). Anti-35K antibodies also cross-reacted with one or more of the SmD proteins or streptavidin; however, affinity purifi- cation of these antibodies abolished this cross-reaction (data not shown). Interestingly, antibodies against the 48K and 20K proteins recognized multiple, slightly higher mo- lecular-weight bands, suggesting that ...
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... proteins recognized multiple, slightly higher mo- lecular-weight bands, suggesting that posttranslationally modified forms of these proteins might be present. Consis- tent with our MS data, immunoblotting confirmed the ab- sence of the 65K, 31K, and 20K proteins in the U11-en- riched snRNPs, and the presence of the 59K, 48K, 35K, and 25K proteins (Fig. 6A, odd lanes). In the U11-enriched 12S snRNPs, the majority of the 48K protein recognized by anti-48K antibodies migrated as a single lower-molecular- weight band, suggesting that potentially modified forms of this protein may be present primarily in the 18S U11/U12 ...
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... and/or 12S U11 snRNP, immunoprecipitation studies were performed with affinity-purified antibodies against the 65K, 59K, 48K, 35K, 31K, 25K, or 20K proteins, and 18S or 12S glycerol gradient fractions containing spliceosomal snRNPs (Fig. 1). Coprecipitated snRNAs were subsequently separated by de- naturing PAGE and visualized by Northern blotting (Fig. 6B). Antibodies against the 31K, 48K, or 65K protein failed to precipitate U11/U12 or U11 snRNPs (data not shown), which may indicate that the epitopes recognized by these antibodies are not accessible in these particles. In contrast, precipitation of U11/U12 and U11 snRNPs was observed with antibodies against the 35K and 25K proteins, ...
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Using an in vitro system in which a 5' splice site (5'SS) RNA oligo (AAG decreases GUAAGUAdT) is capable of inducing formation of U2/U4/U5/U6 snRNP complex we show that this oligo specifically binds to U4/U5/U6 snRNP and cross-links to U6 snRNA in the absence of U2 snRNP. Moreover, 5'SS RNA oligo bound to U4/U5/U6 snRNP is chased to U2/U4/U5/U6 snR...
Genetic predisposition to type 1 diabetes (T1D) has been associated with a chromosome 11 locus centered on the proinsulin gene (INS) and with differential steady-state levels of INS RNA from T1D-predisposing and -protective haplotypes. Here, we show that the haplotype-specific expression is determined by INS variants that control the splicing effic...
Pre-mRNA splicing is catalyzed by the spliceosome, and its control is essential for correct gene expression. While splicing repressors typically interfere with transcript recognition by spliceosomal components, the yeast protein L30 blocks spliceosomal rearrangements required for the engagement of U2 snRNP (small ribonucleoprotein particle) to its...
Citations
... The C-terminal RNA recognition motif (RRM) of U11 / U12-65K binds the 3 -terminal stem-loop of U12 snRNA ( 26 ), but can also interact with the 3 -terminal stem-loop of the U6atac snRNA ( 27 ). ZRSR2, a component of the U11 / U12 di-snRNP responsible for 3 splice site recognition ( 20 ), has also been shown to almost exclusively affect splicing of U12-type introns in the cell ( 28 ), even though it may also have a separate role in the major spliceosome ( 29 ). ...
Here, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of U11/U12-65K, a known unique component of the U11/U12 di-snRNP. Both proteins use their highly similar C-terminal RRMs to bind to 3′-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with U11/U12-65K, which uses its N-terminal region to interact with U11 snRNP during intron recognition. Finally, while RBM41 knockout cells are viable, they show alterations in U12-type 3′ splice site usage. Together, our results highlight the role of the 3′-terminal stem-loop of U12 snRNA as a dynamic binding platform for the U11/U12-65K and RBM41 proteins, which function at distinct stages of the assembly/disassembly cycle.
... Since ZRSR2 was copurified with the 18S U11-U12 di-snRNP that recognizes both the 5 splice site and the BPS of a U12-type intron ( 34 ), ZRSR2 might contact the 5 splice site of U12-type introns. To determine whether ZRSR2 contacts 5 splice sites, we plotted ZRSR2 CLIP read coverage relative to 5 splice sites of U12-type introns. ...
... The loss of ZRSR2 activity in cell lines leads to defects confined to U12-type intron splicing, suggesting that ZRSR2 plays either a limited or redundant role in in vivo splicing of U2-type introns ( 14 ). Furthermore, ZRSR2 has been found in the human 18S U11 / U12 di-snRNP but not in the U2-dependent spliceosome ( 34 ), consistent with a limited, if any, role of ZRSR2 in splicing of U2-type introns. ...
Recurring mutations in genes encoding 3′ splice-site recognition proteins, U2AF1 and ZRSR2 are associated with human cancers. Here, we determined binding sites of the proteins to reveal that U2-type and U12-type splice sites are recognized by U2AF1 and ZRSR2, respectively. However, some sites are spliced by both the U2-type and U12-type spliceosomes, indicating that well-conserved consensus motifs in some U12-type introns could be recognized by the U2-type spliceosome. Nucleotides flanking splice sites of U12-type introns are different from those flanking U2-type introns. Remarkably, the AG dinucleotide at the positions −1 and −2 of 5′ splice sites of U12-type introns with GT-AG termini is not present. AG next to 5′ splice site introduced by a single nucleotide substitution at the −2 position could convert a U12-type splice site to a U2-type site. The class switch of introns by a single mutation and the bias against G at the −1 position of U12-type 5′ splice site support the notion that the identities of nucleotides in exonic regions adjacent to splice sites are fine-tuned to avoid recognition by the U2-type spliceosome. These findings may shed light on the mechanism of selectivity in U12-type intron splicing and the mutations that affect splicing.
... Together, our results highlight the role 3'-terminal stem-loop of U12 snRNA as a dynamic binding platform for the paralogous U11/U12-65K and RBM41 proteins, which function at distinct stages of minor spliceosome assembly/disassembly cycle. : bioRxiv preprint 4 (Will et al., 2004), has also shown to almost exclusively affect splicing of U12-type introns in the cell (Madan et al., 2015), even though it may also have a separate role in the major spliceosome (Shen et al., 2010). The notion that specific protein components of the minor spliceosome are needed only during the intron recognition phase and not in the later assembly steps has been challenged only very recently. ...
... Both the major and the minor spliceosome contain four unique small nuclear RNAs (snRNAs) and a number of unique protein components that are not found in the other spliceosome, while the majority of proteins are likely to be shared (Bai et al., 2021;Schneider et al., 2002;Will et al., 2004;Will et al., 1999). The first set of minor spliceosome-specific proteins (20K, 25K, 31K, 35K, 48K, 59K, 65K) were identified over 20 years ago by affinity purification and mass spectrometry analysis of the U11/U12 di-snRNP and U11 mono-snRNP fractions (Will et al., 2004;Will et al., 1999). ...
... Both the major and the minor spliceosome contain four unique small nuclear RNAs (snRNAs) and a number of unique protein components that are not found in the other spliceosome, while the majority of proteins are likely to be shared (Bai et al., 2021;Schneider et al., 2002;Will et al., 2004;Will et al., 1999). The first set of minor spliceosome-specific proteins (20K, 25K, 31K, 35K, 48K, 59K, 65K) were identified over 20 years ago by affinity purification and mass spectrometry analysis of the U11/U12 di-snRNP and U11 mono-snRNP fractions (Will et al., 2004;Will et al., 1999). Of these, U11/U12-65K, U11-59K and U11-48K form a chain of interactions connecting the U11 and U12 mono-snRNPs into a di-snRNP (Benecke et al., 2005;Turunen et al., 2008) and are essential for the stability of the di-snRNP (Argente et al., 2014;Norppa et al., 2018;Turunen et al., 2008). ...
In this work, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of the U11/U12-65K protein, a known unique component of the minor spliceosome that functions during the early steps of minor intron recognition as a component of the U11/U12 di-snRNP. We show that both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with the U11/U12-65K protein, which uses the N-terminal region to interact with U11 snRNP during the intron recognition step. Finally, we show that while RBM41 knockout cells are viable, they show alterations in the splicing of U12-type introns, particularly differential U12-type 3' splice site usage. Together, our results highlight the role 3'-terminal stem-loop of U12 snRNA as a dynamic binding platform for the paralogous U11/U12-65K and RBM41 proteins, which function at distinct stages of minor spliceosome assembly/disassembly cycle.
... The U11/U12 complex consists of at least eight U11/U12 RNP specific proteins and is involved in alternative splicing [35,47]. Members of the complex include hPrp43 (DHX15), RNPC-3, PDCD7, snRNP48, snRNP35, ZCRB1, snRNP25, and ZMAT5 (see Table 3). ...
Anti-nuclear (ANA) are present in approximately 90% of systemic sclerosis (SSc) patients and are key biomarkers in supporting the diagnosis and determining the prognosis of this disease. In addition to the classification criteria autoantibodies for SSc [i.e., anti-centromere, anti-topoisomerase I (Scl-70), anti-RNA polymerase III], other autoantibodies have been associated with important SSc phenotypes. Among them, anti-U11/U12 ribonucleoprotein (RNP) antibodies, also known as anti-RNPC-3, were first reported in a patient with SSc, but very little is known about their association and clinical utility. The U11/U12 RNP macromolecular complex consists of several proteins involved in alternative mRNA splicing. More recent studies demonstrated associations of anti-anti-U11/U12 antibodies with SSc and severe pulmonary fibrosis as well as with moderate to severe gastrointestinal dysmotility. Lastly, anti-U11/U12 autoantibodies have been strongly associated with malignancy in SSc patients. Here, we aimed to summarize the knowledge of anti-U11/U12/RNPC-3 antibodies in SSc, including their seroclinical associations in a narrative literature review.
... SNRPD2 is one of the 141 proteins considered core components of the human spliceosome based on their high abundance or function [33,34]. It is found in the precatalytic spliceosome B complex, the activated spliceosome C complexes, and the minor U12 spliceosome [35][36][37]. This protein plays a role in the appropriate cohesion of sister chromatids and cell proliferation [38] and in the nuclear retention of lncRNAs [39]. ...
... Ponceau S red staining is shown in the lower panel. M, molecular weight markers (size of bands in kDa: 250, 150, 100, 75,50,37,25,20). ...
... Different versions of GST-SNRPD2 used are indicated: wild-type (wt), point mutants K85A, K92A and K85A/K92A and deletion mutant Δ84-92. M, molecular weight markers (size of bands in kDa: 250, 150, 100, 75,50,37,25,20). (C) Quantification of ubiquitinated bands from three independent experiments. ...
SlrP is a protein with E3 ubiquitin ligase activity that is translocated by Salmonella enterica serovar Typhimurium into eukaryotic host cells through a type III secretion system. A yeast two-hybrid screen was performed to find new human partners for this protein. Among the interacting proteins identified by this screen was SNRPD2, a core component of the spliceosome. In vitro ubiquitination assays demonstrated that SNRPD2 is a substrate for the catalytic activity of SlrP, but not for other members of the NEL family of E3 ubiquitin ligases, SspH1 and SspH2. The lysine residues modified by this activity were identified by mass spectrometry. The identification of a new ubiquitination target for SlrP is a relevant contribution to the understanding of the role of this Salmonella effector.
... ZMAT5 and SCNM1 are part of the U1-type zinc finger family. The equivalent of ZMAT5 in the major spliceosome is SNRPC (Will et al. 2004) and SCNM1 functions as a combination of SF3A2 and SF3A3 (Bai et al. 2021). Although the phylogenetic tree of this family is unresolved, it is likely that these major and minor spliceosome equivalents are sister paralogs. ...
Eukaryotic genes are characterised by the presence of introns that are removed from the pre-mRNA by the spliceosome. This ribonucleoprotein complex is comprised of multiple RNA molecules and over a hundred proteins, which makes it one of the most complex molecular machines that originated during the prokaryote-to-eukaryote transition. Previous work has established that these introns and the spliceosomal core originated from self-splicing introns in prokaryotes. Yet it remains largely elusive how the spliceosomal core expanded by recruiting many additional proteins. In this study we use phylogenetic analyses to infer the evolutionary history of the 145 proteins that we could trace back to the spliceosome in the last eukaryotic common ancestor (LECA). We found that an overabundance of proteins derived from ribosome-related processes were added to the prokaryote-derived core. Extensive duplications of these proteins substantially increased the complexity of the emerging spliceosome. By comparing the intron positions between spliceosomal paralogs, we infer that most spliceosomal complexity postdates the spread of introns through the proto-eukaryotic genome. The reconstruction of early spliceosomal evolution provides insight into the driving forces behind the emergence of complexes with many proteins during eukaryogenesis.
... There are two RRM domains present in SF3B4, as shown in Fig. 2. SF3B4 is a major subunit of the U2-type of the spliceosome. SF3B4 is also associated with minor spliceosome responsible for pre-mRNA splicing of scarce introns [56]. Both SF3B4 upregulations, as well as amplification, were observed in pre-cancerous tissues of HCC. ...
... SNRPB plays crucial role in pre-mRNA splicing as core component of the SMN-Sm complex, minor U12 spliceosome [56], U1, U2, U4 and U5 small nuclear ribonucleoproteins (snRNPs) [93][94][95][96], pre-catalytic spliceosome complex B, and activated spliceosome C complexes [46,93,97] mediates the spliceosomal snRNP assembly. It is also a component of the U7 snRNP which is involved in histone pre-mRNA 3′-end processing [98], and consists of one SM domain. ...
RNA splicing is the fundamental process that brings diversity at the transcriptome and proteome levels. The spliceosome complex regulates minor and major processes of RNA splicing. Aberrant regulation is often associated with different diseases, including diabetes, stroke, hypertension, and cancer. In the majority of cancers, dysregulated alternative RNA splicing (ARS) events directly affect tumor progression, invasiveness, and often lead to poor survival of the patients. Alike the rest of the gastrointestinal malignancies, in hepatocellular carcinoma (HCC), which alone contributes to ~ 75% of the liver cancers, a large number of ARS events have been observed, including intron retention, exon skipping, presence of alternative 3′-splice site (3′SS), and alternative 5′-splice site (5′SS). These events are reported in spliceosome and non-spliceosome complexes genes. Molecules such as MCL1, Bcl-X, and BCL2 in different isoforms can behave as anti-apoptotic or pro-apoptotic, making the spliceosome complex a dual-edged sword. The anti-apoptotic isoforms of such molecules bring in resistance to chemotherapy or cornerstone drugs. However, in contrast, multiple malignant tumors, including HCC that target the pro-apoptotic favoring isoforms/variants favor apoptotic induction and make chemotherapy effective. Herein, we discuss different splicing events, aberrations, and antisense oligonucleotides (ASOs) in modulating RNA splicing in HCC tumorigenesis with a possible therapeutic outcome.
... The minor spliceosome is comprised of five snRNAs (U11, U12, U4atac, U5, U6atac) and seven associated proteins (ZMAT5/20K, SNRNP25/25K, ZCRB1/31K, SNRNP35/35K, SNRNP48/48K, PDCD7/59K, and RNPC3/65K) (Fig. 6A). These components are exclusive to the minor spliceosome and are not found in the major spliceosome, except for the snRNA U5 that is common to both spliceosomes (Will et al., 2004). To investigate whether human heart disease may be associated with perturbations in the activity of the minor spliceosome, we compared the expression of these components in the 100 non-diseased hearts with that of 128 dilated cardiomyopathy (DCM) hearts (Heinig et al., 2017). ...
Eukaryotic genomes contain a tiny subset of ‘minor class’ introns with unique sequence elements that require their own splicing machinery. These minor introns are present in certain gene families with specific functions, such as voltage-gated sodium and voltage-gated calcium channels. Removal of minor introns by the minor spliceosome has been proposed as a post-transcriptional regulatory layer, which remains unexplored in the heart. Here, we investigate whether the minor spliceosome regulates electrophysiological properties of cardiomyocytes by knocking-down the essential minor spliceosome component U6atac in neonatal rat ventricular myocytes. Loss of U6atac led to robust minor intron retention within Scn5a and Cacna1c, resulting in reduced protein levels of Nav1.5 and Cav1.2. Functional consequences were studied through path-clamp analysis, and revealed reduced sodium and L-type calcium currents after loss of U6atac. In conclusion, minor intron splicing modulates voltage-dependent ion channel expression and function in cardiomyocytes. This may be of particular relevance in situations in which minor splicing activity changes, such as in genetic diseases affecting minor spliceosome components, or in acquired diseases in which minor spliceosome components are dysregulated, such as heart failure.
... CWC22 expression levels were associated with colon cancer and its silencing led to increased p53 levels 38,39 . SNRPD3 is also part of the spliceosome complex 40 . It has been found to have a regulatory effect on p53 expression in non-small cell lung cancer. ...
FRG1 has a role in tumorigenesis and angiogenesis. Our preliminary analysis showed that FRG1 mRNA expression is associated with overall survival (OS) in certain cancers, but the effect varies. In cervix and gastric cancers, we found a clear difference in the OS between the low and high FRG1 mRNA expression groups, but the difference was not prominent in breast, lung, and liver cancers. We hypothesized that FRG1 expression level could affect the functionality of the correlated genes or vice versa, which might mask the effect of a single gene on the OS analysis in cancer patients. We used the multivariate Cox regression, risk score, and Kaplan Meier analyses to determine OS in a multigene model. STRING, Cytoscape, HIPPIE, Gene Ontology, and DAVID (KEGG) were used to deduce FRG1 associated pathways. In breast, lung, and liver cancers, we found a distinct difference in the OS between the low and high FRG1 mRNA expression groups in the multigene model, suggesting an independent role of FRG1 in survival. Risk scores were calculated based upon regression coefficients in the multigene model. Low and high-risk score groups showed a significant difference in the FRG1 mRNA expression level and OS. HPF1, RPL34, and EXOSC9 were the most common genes present in FRG1 associated pathways across the cancer types. Validation of the effect of FRG1 mRNA expression level on these genes by qRT-PCR supports that FRG1 might be an upstream regulator of their expression. These genes may have multiple regulators, which also affect their expression, leading to the masking effect in the survival analysis. In conclusion, our study highlights the role of FRG1 in the survivability of cancer patients in tissue-specific manner and the use of multigene models in prognosis.
... Interestingly, SF3B1 participates also in the minor U12-dependent spliceosome (Figure 2A, [174]). However, the impact of hotspot SF3B1 mutations on minor intron splicing has not been investigated yet. ...
Myeloid neoplasms encompass a very heterogeneous family of diseases characterized by the failure of the molecular mechanisms that ensure a balanced equilibrium between hematopoietic stem cells (HSCs) self-renewal and the proper production of differentiated cells. The origin of the driver mutations leading to preleukemia can be traced back to HSC/progenitor cells. Many properties typical to normal HSCs are exploited by leukemic stem cells (LSCs) to their advantage, leading to the emergence of a clonal population that can eventually progress to leukemia with variable latency and evolution. In fact, different subclones might in turn develop from the original malignant clone through accumulation of additional mutations, increasing their competitive fitness. This process ultimately leads to a complex cancer architecture where a mosaic of cellular clones—each carrying a unique set of mutations—coexists. The repertoire of genes whose mutations contribute to the progression toward leukemogenesis is broad. It encompasses genes involved in different cellular processes, including transcriptional regulation, epigenetics (DNA and histones modifications), DNA damage signaling and repair, chromosome segregation and replication (cohesin complex), RNA splicing, and signal transduction. Among these many players, transcription factors, RNA splicing proteins, and deubiquitinating enzymes are emerging as potential targets for therapeutic intervention.
