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SL4 of U1 snRNA is important for U1 function. (A) Schematic of the secondary structures of SL4 region of the wild-type and mutant U1 snRNAs. Structures predicted to have the lowest DG are shown (Zuker 2003). (B) Primer extension analysis of the Dup51 minigene transcripts after cotransfection with control or U1 plasmids. The mRNA products are indicated at the right and quantified in the graph below. (C) Primer extension analysis with oligonucleotide U1 7-26 showing expression of the endogenous wild-type U1 and variant U1-5a snRNAs.
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The pairing of 5' and 3' splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. H...
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... U1 snRNA in higher eukaryotes folds into a struc- ture containing four stem-loops, with SL4 at the 39 terminus downstream from the Sm protein-binding site ( Burge et al. 1999). SL4 consists of two G-C-rich stems split by a pyrimidine-rich internal loop and capped by a UUCG tetraloop ( Fig. 2A). To examine possible re- quirements of SL4 in U1 function, we introduced changes in this region of the U1-5a construct and tested their effect on the rescue of the 59 splice site mutation in the Dup51p reporter (Fig. 2A). The pyrimidines in the bulge and the tetraloop were changed to adenosine in mutant U1-5aM3. The G-C base pairs were ...
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... Burge et al. 1999). SL4 consists of two G-C-rich stems split by a pyrimidine-rich internal loop and capped by a UUCG tetraloop ( Fig. 2A). To examine possible re- quirements of SL4 in U1 function, we introduced changes in this region of the U1-5a construct and tested their effect on the rescue of the 59 splice site mutation in the Dup51p reporter (Fig. 2A). The pyrimidines in the bulge and the tetraloop were changed to adenosine in mutant U1-5aM3. The G-C base pairs were changed to A-U in either the upper stem (M10b) or the lower stem (M10a) or both stems (M10). In mutants M10c, M10d, and M10e, the strands of the stems were swapped. In M10u, G-C base pairs were changed to C-G base pairs ...
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... effect of the SL4 mutations on U1 snRNP activity was assayed by cotransfection of the U1-5a constructs with the Dup51p reporter. The U1-5a variants carrying SL4 mutations were compared with U1-5a carrying the wild-type SL4 for their ability to restore exon 2 splicing (Fig. 2B, cf. lanes 4-20 and lane 3). SL4 sequence muta- tions that affect U1 function will compromise the ability of the U1-5a construct to rescue exon 2 inclusion. Many changes in SL4 did not affect U1-5a activity in restoring exon 2 splicing, indicating lack of a significant role for those SL4 nucleotides. Notably, changing the pyrimidine ...
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... bulge regions and tetra- loop to adenosines did not affect the ability of U1 to restore exon 2 splicing ( Fig From the suppressor U1 analysis, we found that U1 function was most affected by mutations that alter the strength of base-pairing in the lower stem of SL4. Chang- ing the G-C-rich lower stem to an A-U base-paired stem (M10 and M10a) (Fig. 2B, lanes 5,6) led to loss of exon 2 splicing. G-C-to-A-U changes in the upper stem alone had minimal effect (M10b) (Fig. 2B, lane 7). The largest effects were observed when the majority of base pairs in the lower stem were disrupted (M10g, M10h, M10q, M10r, and M10m) (Fig. 2B, lanes 13,14,16,17,20). This analysis shows that the lower G-C-rich stem ...
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... suppressor U1 analysis, we found that U1 function was most affected by mutations that alter the strength of base-pairing in the lower stem of SL4. Chang- ing the G-C-rich lower stem to an A-U base-paired stem (M10 and M10a) (Fig. 2B, lanes 5,6) led to loss of exon 2 splicing. G-C-to-A-U changes in the upper stem alone had minimal effect (M10b) (Fig. 2B, lane 7). The largest effects were observed when the majority of base pairs in the lower stem were disrupted (M10g, M10h, M10q, M10r, and M10m) (Fig. 2B, lanes 13,14,16,17,20). This analysis shows that the lower G-C-rich stem of SL4 plays an important role in U1 snRNP ...
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... expression of all mutant U1-5a snRNAs was confirmed by primer extension (Fig. 2C). Quantification of the mutant snRNA expression indicated that at the observed levels, the exogenous U1-5a snRNAs were not limiting for the splicing of the reporter pre-mRNA (see the Materials and Methods; Supplemental Fig. S1). The . Suppressor U1 snRNAs can rescue splic- ing. (A) Schematic representation of three-exon/two- intron ...
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... comparison of U1-SL4 sequences from humans, Drosophila, Caenorhabditis elegans, and S. pombe shows significant differences in their sequences and structures (Supplemental Fig. S2A). Human SL4 shares 68%, 59%, and 19% sequence identity with the Dro- sophila, C. elegans, and S. pombe RNAs, respectively, although all of these SL4 sequences can be folded into stem-loop structures (Supplemental Fig. S2B). In C. elegans and S. pombe, the stem lacks the pyrimi- dine-containing internal loop, and in Drosophila, there is ...
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... Drosophila, Caenorhabditis elegans, and S. pombe shows significant differences in their sequences and structures (Supplemental Fig. S2A). Human SL4 shares 68%, 59%, and 19% sequence identity with the Dro- sophila, C. elegans, and S. pombe RNAs, respectively, although all of these SL4 sequences can be folded into stem-loop structures (Supplemental Fig. S2B). In C. elegans and S. pombe, the stem lacks the pyrimi- dine-containing internal loop, and in Drosophila, there is a single bulged adenosine. In S. pombe, the loop is bigger than the tetraloop found in other organisms, and the stem is shorter. In all species, the lower portion of the terminal stem is highly G-C-rich. Note that the ...
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... and U1-5aM10t constructs that eliminate the internal loop separating the upper and lower stems (Supplemental Fig. S1B). We also made chimeric U1-5a constructs carrying the Drosophila (Dm), C. elegans (Ce), and S. pombe (Sp) sequences in place of the human SL4. All of these constructs were active in the U1 complementation assay (Supplemental Fig. S2C, lanes 4-8). The activity of the U1-5aSL4Sp construct showed that the size of the SL4 loop is not critical for U1 function. The G-C base pairs at the base of SL4 that were found to be important for the human U1 function are present in all of these constructs. Taken together, these experiments indicate that the G-C base pairs in the ...
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... with increasing concentrations of NaCl and then used for RNA affinity purification using biotinylated wild-type and mutant U1-SL4 RNAs (Fig. 3A). The bound complexes were analyzed for snRNAs and U2 snRNP- specific proteins. The snRNA analysis showed binding of the U2 snRNA to the wild-type U1-SL4 in extract in standard splicing conditions (Fig. 5B, lane 2). Preincuba- tion of the extract with 250 mM or higher salt concen- trations led to loss of U2 snRNA binding (Fig. 5B, lanes 3- 5). The U2 snRNA did not bind to the mutant SL4 complex in any condition (Fig. 5B, lanes ...
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... seen previously, immunoblot analysis of proteins bound to wild-type U1-SL4 in splicing conditions con- firmed the binding of U2 snRNP proteins SF3A1, SF3A2, SF3A3, SF3B1, and U2B0 (Fig. 5C, lane 2). In contrast, the U1 snRNP-specific protein U1C did not bind to SL4. After preincubation of the extract with NaCl, binding of SF3A2 (66 kDa), SF3B1 and the core U2 protein U2B0 were lost (Fig. 5C, lanes 3-5). Interestingly, although the amounts of SF3A1 (120 kDa) and SF3A3 (60 kDa) proteins bound to the wild-type U1-SL4 decreased after ...
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... the U1 snRNP-specific protein U1C did not bind to SL4. After preincubation of the extract with NaCl, binding of SF3A2 (66 kDa), SF3B1 and the core U2 protein U2B0 were lost (Fig. 5C, lanes 3-5). Interestingly, although the amounts of SF3A1 (120 kDa) and SF3A3 (60 kDa) proteins bound to the wild-type U1-SL4 decreased after salt preincubation (Fig. 5C, lanes 2-5), the interaction of these proteins was more resistant to NaCl, and substantial amounts of each of these proteins remained bound at higher salt concen- trations. The mutant SL4 did not bind SF3B1, U2B0, SF3A1, and SF3A2 (Fig. 5C, lanes 6-9). The SF3A3 protein did bind to the mutant SL4 but at lower levels than to wild-type. These ...
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... an ;55-kDa protein to the wild-type SL4 (Fig. 6A, lane 1). The ;55-kDa protein cross-links to both the wild-type and mutant SL4 RNAs and is pre- sumably PTBP1 (see below). The 120-kDa protein did not cross-link efficiently to the mutant SL4 RNA (Fig. 6A, lanes 5-8). Most interestingly, the anti-SF3A1 antibody (Fig. 6A, lane 3), but not the U1 70k (Fig. 6A, lane 2) or SF3A3 (Fig. 6A, lane 4) antibody, significantly enriched the ;120-kDa protein. Thus, the binding of U2 snRNP to the wild-type U1-SL4 likely occurs through direct contact with the SF3A1 ...
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... cross-linked, and immunoprecipitated using an anti-SF3A1 antibody that was prebound to g-bind beads. The bound complexes were digested with SDS/proteinase K, and the total RNA was extracted, labeled with 32 P-pCp, and analyzed for the presence of pre-mRNA and snRNAs (Fig. 6B). Equivalent gradient fractions from control reactions lacking pre-mRNA (Fig. 6B, lanes 2-4) were treated identically to the fractions from pre-mRNA-containing reactions (Fig. 6B, lanes ...
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... ). (Lane 1) Total RNA from nuclear extract was used as a marker for the U snRNAs. The positions of the U snRNAs and pre-mRNA are indicated. using RNA affinity chromatography and immunoblot analysis. Synthetic biotinylated SL4 RNAs-including the wild-type, the M10 and M10h mutants, and the Drosophila SL4 ( Fig. 2; Supplemental Fig. S2)-were incubated under splicing conditions in HeLa nuclear ex- tract, and the bound proteins were isolated on Neutravidin beads as described above. The binding of SF3A1 and PTBP1 was assessed by immunoblot (Fig. 7A). The ratio of protein bound to each SL4 variant to that bound to the wild-type human SL4 was determined. Interestingly, ...
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... but bound at higher levels to the Drosophila SL4 (Fig. 7B, cf. lanes 3,4 and lane 5). Thus, the binding of SF3A1 correlated with the presence of the G-C-rich stem structures and not the presence of the bulged nucleotides, as was seen for U1 activity in the in vivo assays. In contrast, PTBP1 bound well to both the wild-type and M10 mutant RNAs (Fig. 7B, lanes 2,3). Both of these RNAs contain the bulged pyrimidines that were shown previously to bind PTBP1 ( Sharma et al. 2011). The M10h mutant, which contains these nucleotides but not in a bulged structure, showed moderate binding to PTBP1 (Fig. 7B, lane 4). In contrast, PTBP1 bound very poorly to the SL4-Dm RNA that lacks the pyrimidine bulge ...
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... with the ratios ranging from ;0.6 to 1.2 for different experiments (Supplemental Fig. S1). These high accumulation levels did not apparently result in increased activity, as the level of exon 2 inclusion with coexpression of M10f (;82%) (Fig. 2B, lane 12) and M10p (;72%) (Fig. 2B, lane 15) was still lower than that observed for U1-5a (;94%) (Fig. 2B, lane 3). Some other U1 mutants (such as M3, M10d, and M10e) that have expression ratios in the lower range (0.11-0.17) had exon 2 inclusion levels of $90%. These data indicate that at the observed snRNA expression levels, the exogenous U1-5a snRNAs were not limiting for the splicing of the reporter pre-mRNAs. Changes in the reporter splicing ...
Citations
... With the exception of a crosslink between SF3A1 and PRPF40A, crosslinks between U1 and U2 snRNP proteins are not observed, supporting the conclusion that U2 and U1 do not directly contact one another in cross-exon complexes, as previously proposed. The U1 snRNA stem-loop 4 was previously shown to interact with the U2 SF3A1 protein during the early stages of cross-intron spliceosome assembly 47 . However, it is not clear whether this RNA-protein interaction, which would directly connect U1 and U2 snRNP, contributes to the molecular bridge linking U1 and U2 in cross-exon complexes. ...
Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron¹. Alternatively, it can occur through an exon-defined pathway2–5, whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5′ splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal B complex6,7. Exon definition promotes the splicing of upstream introns2,8,9 and plays a key part in alternative splicing regulation10–16. However, the three-dimensional structure of exon-defined spliceosomal complexes and the molecular mechanism of the conversion from a CE-organized to a CI-organized spliceosome, a pre-requisite for splicing catalysis, remain poorly understood. Here cryo-electron microscopy analyses of human CE pre-B complex and B-like complexes reveal extensive structural similarities with their CI counterparts. The results indicate that the CE and CI spliceosome assembly pathways converge already at the pre-B stage. Add-back experiments using purified CE pre-B complexes, coupled with cryo-electron microscopy, elucidate the order of the extensive remodelling events that accompany the formation of B complexes and B-like complexes. The molecular triggers and roles of B-specific proteins in these rearrangements are also identified. We show that CE pre-B complexes can productively bind in trans to a U1 snRNP-bound 5′ splice site. Together, our studies provide new mechanistic insights into the CE to CI switch during spliceosome assembly and its effect on pre-mRNA splice site pairing at this stage.
... There are several interactions during the early stages of spliceosome assembly, which bridge the 5′ and 3′SS, both across the intervening intron and exon. For example, the stem loop 4 of the U1 snRNA (5′SS) is recognized by the UBL-like domain of SF3A1 (U2 snRNP component in 3′SS) 21,22 ; the UAP56 (RNA helicase) binds to the U1 snRNA stem loop 3 enhancing complex A formation 23 ; the SR proteins are thought to mediate U1 and U2 snRNPs bridging through their serine-arginine motifs [24][25][26][27] ; and recently, FUBP1 was identified as a core splicing factor that binds upstream of the BPS but also mediates interactions with the U1 snRNP 28 . The splicing factor PRPF40A was proposed to mediate a bridging connection already in complex E (prior binding of U2 snRNP) by the simultaneous recognition of U1 snRNP at the 5′ SS and SF1 at the BPS 29,30 . ...
PRPF40A plays an important role in the regulation of pre-mRNA splicing by mediating protein-protein interactions in the early steps of spliceosome assembly. By binding to proteins at the 5´ and 3´ splice sites, PRPF40A promotes spliceosome assembly by bridging the recognition of the splices. The PRPF40A WW domains are expected to recognize proline-rich sequences in SF1 and SF3A1 in the early spliceosome complexes E and A, respectively. Here, we combine NMR, SAXS and ITC to determine the structure of the PRPF40A tandem WW domains in solution and characterize the binding specificity and mechanism for proline-rich motifs recognition. Our structure of the PRPF40A WW tandem in complex with a high-affinity SF1 peptide reveals contributions of both WW domains, which also enables tryptophan sandwiching by two proline residues in the ligand. Unexpectedly, a proline-rich motif in the N-terminal region of PRPF40A mediates intramolecular interactions with the WW tandem. Using NMR, ITC, mutational analysis in vitro, and immunoprecipitation experiments in cells, we show that the intramolecular interaction acts as an autoinhibitory filter for proof-reading of high-affinity proline-rich motifs in bona fide PRPF40A binding partners. We propose that similar autoinhibitory mechanisms are present in most WW tandem-containing proteins to enhance binding selectivity and regulation of WW/proline-rich peptide interaction networks.
... The U1 snRNA associates with three U1-specific proteins (U1A, U1-70K and U1C) and seven Sm proteins (SmB/B′,SmC,SmD1,SmD2,SmD3,SmF,and SmG) to form the U1 small nuclear ribonucleoprotein (snRNP) which, via the 5´-region of the snRNA, base pairs to 5´-splice site sequences at exon-intron junctions in nascent pre-mRNAs. During the initial steps of spliceosome assembly, the U1 snRNA also interacts with the RNA helicase U2 associated protein 56 (UAP56, also known as DDX39B) via its stem-loop 3 (SL3) and with the U2 snRNP specific splicing factor 3A1 (SF3A1) via its stem-loop 4 (SL4) (de Vries et al., 2022;Martelly et al., 2021;Sharma, Wongpalee, Vashisht, Wohlschlegel, & Black, 2014). These cross-intron contacts have been found to promote the formation of U1 and U2 containing prespliceosomal A complex and pre-mRNA splicing in vitro . ...
... Although vU1 snRNAs have been shown to be expressed and incorporated into snRNPs and spliceosomes, their ability to facilitate pre-mRNA splicing catalysis has not been tested so far. In this study, we applied a previously established HeLa cell based minigene reporter assay to examine the capacity of vU1 snRNAs to support splicing (Sharma et al., 2014;Wong, Martelly, & Sharma, 2021). The results revealed that even though many of the human vU1s could be exogenously expressed at levels comparable to the canonical U1 snRNA and were enriched in the nucleus, the ability to support pre-mRNA splicing of only a few was appreciable. ...
... The expressed wildtype and mutant snRNAs were found to be processed, localized to the nucleus, and assembled into mature snRNPs. The exogenously expressed wildtype U1 and many of the mutants, carrying changes to SL3 and SL4, were also found to have the capacity to support splicing upon co-expression with a reporter in HeLa cells Sharma et al., 2014). ...
The human U1 snRNA is encoded by a multigene family consisting of transcribed variants and defective pseudogenes. Many variant U1 (vU1) snRNAs have been demonstrated to not only be transcribed but also processed by the addition of a trimethylated guanosine cap, packaged into snRNPs, and assembled into spliceosomes, however, their capacity to facilitate pre-mRNA splicing has, so far, not been tested. A recent systematic analysis of the human snRNA genes identified 178 U1 snRNA genes that are present in the genome as either tandem arrays or single genes on multiple chromosomes. Of these, 15 were found to be expressed in human tissues and cell lines, although at significantly low levels from their endogenous loci, less than 0.001% of the canonical U1 snRNA. In this study, we found that placing the variants in the context of the regulatory elements of the RNU1-1 gene improves expression of many variants to levels comparable to the canonical U1 snRNA. Application of a previously established HeLa cell based minigene reporter assay to examine the capacity of the vU1 snRNAs to support pre-mRNA splicing revealed that even though the exogenously expressed variant snRNAs were enriched in the nucleus, only a few had a measurable effect on splicing.
... Additionally, the N-terminal RRM of U1A binds the SLII [26,27]. Although SLIII and SLIV do not directly bind U1-specific proteins, studies have shown that they interact with the DEAD-Box Polypeptide 39B (DDX39B, also referred to as U2AF65associated protein 56 (UAP56)) and splicing factor 3a subunit 1 (SF3A1), respectively [28,29]. These interactions facilitate the transition from CC to pre-spliceosome complex formation and subsequently contribute to pre-spliceosome complex stabilization [29]. ...
Over the last decade, our understanding of spliceosome structure and function has significantly improved, refining the study of the impact of dysregulated splicing on human disease. As a result, targeted splicing therapeutics have been developed, treating various diseases including spinal muscular atrophy and Duchenne muscular dystrophy. These advancements are very promising and emphasize the critical role of proper splicing in maintaining human health. Herein, we provide an overview of the current information on the composition and assembly of early splicing complexes-commitment complex and pre-spliceosome-and their association with human disease.
... Examples of such connections involve interactions between U1 snRNP and the 3′ and 5′ domains of U2 snRNP 34 ; the interaction of stem-loop IV of U1 snRNA (which is essential for splicing) with a non-canonical RNA binding domain in the U2 snRNP protein SF3A1 (refs. 96,97); the interaction of stem-loop III of U1 snRNA with the U2AF-associated RNA helicase UAP56 (ref. 98); and the interaction of SF1 with the U1 snRNP-associated protein Prp40p in yeast 99,100 (Fig. 2). ...
The removal of introns from mRNA precursors and its regulation by alternative splicing are key for eukaryotic gene expression and cellular function, as evidenced by the numerous pathologies induced or modified by splicing alterations. Major recent advances have been made in understanding the structures and functions of the splicing machinery, in the description and classification of physiological and pathological isoforms and in the development of the first therapies for genetic diseases based on modulation of splicing. Here, we review this progress and discuss important remaining challenges, including predicting splice sites from genomic sequences, understanding the variety of molecular mechanisms and logic of splicing regulation, and harnessing this knowledge for probing gene function and disease aetiology and for the design of novel therapeutic approaches. Alternative splicing of pre-mRNAs is key for cellular function and underpins the aetiology of numerous diseases. Here, we review major advances in understanding the structures and functions of the splicing machinery and its regulation, and in harnessing this knowledge for the design of novel therapies.
... SF3A1, as an important member of SF3a, plays an important role in selective splicing. Sha et al. found that stem-loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-speci c SF3A1 protein during spliceosome assembly [29]. In vivo experiments showed that SF3A1 interacted with the P2X6 subunit, ultimately leading to a decrease in the splicing activity of mRNA [30]. ...
Background: Splicing factor 3A subunit 1 (SF3A1)-related pathways involve gene expression and mRNA splicing. To date, no direct association of SF3A1 with tuberculosis or similar infectious diseases has been reported in the literature.
Methods: A case‒control study was conducted in 1137 patients with tuberculosis (558 with severe tuberculosis and 579 with mild tuberculosis) and 581 healthy controls.
Whole blood DNA was extracted from all patients. Four tag polymorphisms (rs2074733, rs10376, rs117435254, and rs2839998) of the SF3A1 gene were selected and genotyped using a SNPscan Kit (Cat#: G0104, Genesky Biotechnologies Inc., Shanghai, China).
Results: The rs2074733 alleleT and rs10376 alleleA in the SF3A1 gene were associated with increased TB susceptibility after adjusting for age and sex (Pa = 0.036 and 0.048, respectively). No significant association was found between rs117435254 and rs2839998 and tuberculosis infection. In subgroup analyses, we did not find a significant association between SF3A1 gene polymorphisms in patients with mild and severe tuberculosis.
Conclusions: In our study, we found a statistically significant association between the two SNPs (rs2074733 and rs10376) in the SF3A1 gene and susceptibility to tuberculosis infection in a Chinese Han population. To the best of our knowledge, this is the first report on the relationship between the SF3A1 gene and TB.
... The former is likely the preferred way of splicing in species such as drosophila with mostly short introns, and the latter in vertebrates with relatively short exons among long introns [1][2][3][4]. Both scenarios are supported by splice site cross-talks evident by the impact of one splice site defects on the other site across an intron or exon in the genetic or biochemical analysis [1, [5][6][7], or global RNA-Seq analysis of pre-mRNA intermediates in association with intron/exon lengths [8]. The splicing intermediates provide targets for monitoring the splice site usage of endogenous pre-mRNA transcripts [8][9][10][11]. ...
... The long lengths are in contrast to the mostly less-than-0.1kb of intron lengths for intron definition in yeast, worm, or arthropods [2][3][4]8,10]. An explanation for the SS pairing or definition of such long introns perhaps could be helped by the reported 5 SS and 3 SS interactions or recursive splicing [1, [5][6][7][44][45][46]. Moreover, the type II RISE pair's exons are only 124nt on average, but there are also 10 exons longer than 496 nt, at which length exon definition is abolished in vitro [1]. ...
... Potential molecular mechanisms for the regulation of splice site pairings in these cells by KCl depolarization could involve not only the previously identified hnRNP L/LL at the 3 SS of STREX [15,18] but also other partners that allow the 3 SS to cross-talk with the upstream 5 SS. These could include SR proteins, U snRNP components, or cis-acting pre-mRNA sequence motifs as demonstrated previously in other systems [1, [5][6][7]. For the 5-azaC effect on the splice site pairings, the cytidine analog has been known to regulate alternative splicing [49]. ...
Pairing of splice sites across an intron or exon is the central point of intron or exon definition in pre-mRNA splicing with the latter mode proposed for most mammalian exons. However, transcriptome-wide pairing within endogenous transcripts has not been examined for the prevalence of each mode in mammalian cells. Here we report such pairings in rat GH3 pituitary cells by measuring the relative abundance of nuclear RNA-Seq reads at the intron start or end (RISE). Interestingly, RISE indexes are positively correlated between 5′ and 3′ splice sites specifically across introns or exons but inversely correlated with the usage of adjacent exons. Moreover, the ratios between the paired indexes were globally modulated by depolarization, which was disruptible by 5-aza-Cytidine. The nucleotide matrices of the RISE-positive splice sites deviate significantly from the rat consensus, and short introns or exons are enriched with the cross-intron or -exon RISE pairs, respectively. Functionally, the RISE-positive genes cluster for basic cellular processes including RNA binding/splicing, or more specifically, hormone production if regulated by depolarization. Together, the RISE analysis identified the transcriptome-wide regulation of either intron or exon definition between weak splice sites of short introns/exons in mammalian cells. The analysis also provides a way to further track the splicing intermediates and intron/exon definition during the dynamic regulation of alternative splicing by extracellular factors.
... To examine if SRSF1 displacement is in the early spliceosome assembly pathway in cells, we inhibited assembly of U1 snRNP and U2 snRNP by transfecting HeLa cells with a 25-nt long morpholino oligonucleotide that is complementary to the 5 end of U1 snRNA (U1 AMO) or U2 AMO since destabilization of U2 snRNP also inhibits stable recruitment of U1 snRNP without directly impairing the integrity of U1 snRNP (31). Cells were then transfected with AdML minigene and its splicing efficiency was tested by RT PCR: U1 and U2 AMO partially inhibited splicing compared to the scrambled AMO (Supplementary Figure S3E). ...
We recently reported that serine–arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65 and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.
... The rigid stem-loop 4 (SL4) at the 3'-end of U1 snRNA is required for splicing to occur [105]. U1 snRNA SL4 includes a 5 base-pair stem followed by a 2 nt internal loop, a 3 basepair GCG stem, and an apical UUCG structured tetraloop [106,107] (Fig. 3b). ...
... Historically, splicing events were believed to be under the sole dependence of cis-acting regulatory motifs on the pre-mRNA, to which trans-acting splicing factors would bind and tune splice site recognition and spliceosome assembly. Therefore, the discovery of U1 snRNP as a binding platform that tunes 5ʹss definition is a conceptual breakthrough, since it shows that splicing can be modulated through U1-mediated protein-protein or protein-RNA interactions, sometimes independently from cis-acting regulatory sequences [82,87,94,98,100,101,105]. Given the variability of splicing patterns across tissues, between individuals, and along time, the pursuit of a set of formal rules that could predict splicing events from RNA features (i.e. a splicing code) remains largely unsolved. ...
In Eukarya, immature mRNA transcripts (pre-mRNA) often contain coding sequences, or exons, interleaved by non-coding sequences, or introns. Introns are removed upon splicing, and further regulation of the retained exons leads to alternatively spliced mRNA. The splicing reaction requires the stepwise assembly of the spliceosome, a macromolecular machine composed of small nuclear ribonucleoproteins (snRNPs). This review focuses on the early stage of spliceosome assembly, when U1 snRNP defines each intron 5’-splice site (5ʹss) in the pre-mRNA. We first introduce the splicing reaction and the impact of alternative splicing on gene expression regulation. Thereafter, we extensively discuss splicing descriptors that influence the 5ʹss selection by U1 snRNP, such as sequence determinants, and interactions mediated by U1-specific proteins or U1 small nuclear RNA (U1 snRNA). We also include examples of diseases that affect the 5ʹss selection by U1 snRNP, and discuss recent therapeutic advances that manipulate U1 snRNP 5ʹss selectivity with antisense oligonucleotides and small-molecule splicing switches.
... The snRNP was composed of five subunits (U1, U2, U4, U5, and U6) and more than 150 structural proteins [39]. In order to initiate the first step of splicing, it is needed to combine the U1 snRNP and universal protein components with the 5' of mRNA [40]. In our present study, the heterogeneous nuclear ribonucleoprotein U (Hnrnpu) and the U1 small nuclear ribonucleoprotein C (Snrpc) were significantly down-regulated. ...
Background
Bisphenol-A (BPA) has estrogenic activity and adversely affects humans and animals' reproductive systems and functions. There has been a disagreement with the safety of BPA exposure at Tolerable daily intake (TDI) (0.05 mg/kg/d) value and non-observed adverse effect level (5 mg/kg/d). The current study investigated the effects of BPA exposure at various doses starting from Tolerable daily intake (0.05 mg/kg/d) to the lowest observed adverse effect level (50 mg/kg/d) on the testis development in male mice offspring. The BPA exposure lasted for 63 days from pregnancy day 0 of the dams to post-natal day (PND) 45 of the offspring.
Results
The results showed that BPA exposure significantly increased testis (BPA ≥ 20 mg/kg/d) and serum (BPA ≥ 10 mg/kg/d) BPA contents of PND 45 mice. The spermatogenic cells became loose, and the lumen of seminiferous tubules enlarged when BPA exposure at 0.05 mg/kg/d TDI. BPA exposure at a low dose (0.05 mg/kg/d) significantly reduced the expression of Scp3 proteins and elevated sperm abnormality. The significant decrease in Scp3 suggested that BPA inhibits the transformation of spermatogonia into spermatozoa in the testis. The RNA-seq proved that the spliceosome was significantly inhibited in the testes of mice exposed to BPA. According to the RT-qPCR, BPA exposure significantly reduced the expression of Snrpc (BPA ≥ 20 mg/kg/d) and Hnrnpu (BPA ≥ 0.5 mg/kg/d).
Conclusions
This study indicated that long-term BPA exposure at Tolerable daily intake (0.05 mg/kg/d) is not safe because low-dose long-term exposure to BPA inhibits spermatogonial meiosis in mice testis impairs reproductive function in male offspring.
