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Prp5-DPLD motif mutants modulate substrate selectivity of suboptimal branch regions in S. cerevisiae . (A) Schematic of ACT1-CUP1 pre-mRNA, indicating intron mutations at 5 = SS, BS, and 3 = SS used in panel B. (B) Analysis of prp5 -DPLD mutant alleles that alter splicing of branch region mutants. Graphs of maximum copper concentration tolerated (top) and growth on selected copper plates (bottom) are shown. Previously described prp5- N399D and -TAG448 alleles (25) were tested for comparison. prp5 -DPLD mutants improved the copper tolerance of branch region mutants U257C and A258C that decrease pairing with U2 snRNA but do not alter splicing and growth on copper of 5 = SS, 3 = SS, or branch site C or G mutants. The presence of additional base pairs between the branch region and U2 snRNA (25) abrogates the effects of both DPLD mutants and ATPase mutants on the U257C branch region mutation. max, maximum. (C) Additional potential base pairs between U2 snRNA and the intron branch region partially suppress the U257C defect; prp5 alleles provide no additional improvement. (Top) Schematic of base pairing interactions between U2 snRNA and the intron branch region, indicating BS-U257C ACT1-CUP1 reporter mutation, which is improved by prp5 alleles, and (bottom) BS-U257C plus five additional base pairs to U2 snRNA, which is not improved by prp5 alleles, shown in panel B.
Source publication
The assembly of prespliceosomes is responsible for selection of intron sites for splicing. U1 and U2 snRNPs recognize 5′ splice
sites and branch sites, respectively; although there is information regarding the composition of these complexes, little is
known about interaction among the components or between the two snRNPs. Here we describe the prote...
Context in source publication
Context 1
... alter splicing of the wt reporter or of 5=SS, 3=SS, or branch nucleophile mutants, whereas DALD and DPLA mutants did not improve or only slightly improved the splicing of branch region mutants (Fig. 5B, lanes 3 and 5). This pattern of altered splicing is identical to that observed with prp5-ATPase domain mutants (43) (N399D and TAG 448 ; Fig. 5B, lanes 7 and 8), sug- gesting that the DPLD-SF3b interaction is integrally linked with Prp5 ATPase ...
Citations
... A study conducted in Drosophila cells by Liang et al showed that upon depletion of the spliceosomal factors such as U1 and U2 snRNPs, the circRNA expression was elevated with a reduction in the linear mRNA synthesis [85]. U1 snRNP is required for the selection of the pre-mRNA at splice sites and U2 snRNP binds to the branch sites to facilitate canonical splicing [86]. RBPs can promote or repress circRNA biogenesis by binding complementary sequences in the flanking introns or by directly uniting 3' to 5' splice sites. ...
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with limited treatment options. Circular RNAs (circRNAs) have emerged as a novel class of non-coding RNAs with diverse functions in cellular processes. This review paper aims to explore the potential involvement of circRNAs in the pathogenesis of IPF and their diagnostic and therapeutic implications. We begin by providing an overview of the epidemiology and risk factors associated with IPF, followed by a discussion of the pathophysiology underlying this complex disease. Subsequently, we delve into the history, types, biogenesis, and functions of circRNAs and then emphasize their regulatory roles in the pathogenesis of IPF. Furthermore, we examine the current methodologies for detecting circRNAs and explore their diagnostic applications in IPF. Finally, we discuss the potential utility of circRNAs in the treatment of IPF. In conclusion, circRNAs hold great promise as novel biomarkers and therapeutic targets in the management of IPF.
... Three structural motifs at the N-terminal half of PRP5 closely interact with SF3B1, tethering PRP5 to U2 snRNP. These motifs include an elongated helix α1 (residues 152-178), an acidic loop (residues 195-209) and helix α2 (residues 225-243), which harbors the DPLD motif 19,35 (Fig. 2a and Extended Data Fig. 3e-g). ...
Selection of the pre-mRNA branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is crucial to prespliceosome (A complex) assembly. The RNA helicase PRP5 proofreads BS selection but the underlying mechanism remains unclear. Here we report the atomic structures of two sequential complexes leading to prespliceosome assembly: human 17S U2 snRNP and a cross-exon pre-A complex. PRP5 is anchored on 17S U2 snRNP mainly through occupation of the RNA path of SF3B1 by an acidic loop of PRP5; the helicase domain of PRP5 associates with U2 snRNA; the BS-interacting stem-loop (BSL) of U2 snRNA is shielded by TAT-SF1, unable to engage the BS. In the pre-A complex, an initial U2–BS duplex is formed; the translocated helicase domain of PRP5 stays with U2 snRNA and the acidic loop still occupies the RNA path. The pre-A conformation is specifically stabilized by the splicing factors SF1, DNAJC8 and SF3A2. Cancer-derived mutations in SF3B1 damage its association with PRP5, compromising BS proofreading. Together, these findings reveal key insights into prespliceosome assembly and BS selection or proofreading by PRP5.
... RBM39 and its yeast homolog were previously identified in early spliceosome complexes and protein-protein interactions with the U2 snRNP component SF3B1 as well as the U1 snRNP associated factor U1-70K have been described [23][24][25]29,38,39 . Thus, we first aimed to independently verify these interactions using co-immunoprecipitation in HeLa nuclear extracts. ...
Pharmacologic depletion of RNA-binding motif 39 (RBM39) using aryl sulfonamides represents a promising anti-cancer therapy but requires high levels of the adaptor protein DCAF15. Consequently, novel approaches to deplete RBM39 in an DCAF15-independent manner are required. Here, we uncover that RBM39 autoregulates via the inclusion of a poison exon into its own pre-mRNA and identify the cis-acting elements that govern this regulation. We also determine the NMR solution structures of RBM39’s tandem RNA recognition motifs (RRM1 and RRM2) bound to their respective RNA targets, revealing how RRM1 recognises RNA stem loops whereas RRM2 binds specifically to single-stranded N(G/U)NUUUG. Our results support a model where RRM2 selects the 3’-splice site of a poison exon and the RRM3 and RS domain stabilise the U2 snRNP at the branchpoint. Our work provides molecular insights into RBM39-dependent 3’-splice site selection and constitutes a solid basis to design alternative anti-cancer therapies.
... Many IR events have been detected from numerous transcriptomes derived from tumors and cancerous cells in humans (Dvinge & Bradley, 2015) and in response to stress conditions in plants (Filichkin et al., 2018). The RNA binding activity of SR proteins is considered to shape IR, as they bind to sequences within the pre-mRNA in proximity to and within introns ( € Ank€ o et al., 2012;Shao et al., 2012;Wong et al., 2013). While SR proteins play chiefly redundant functions in constitutive splicing, they can also actively select splice sites by modulating their concentration and modularity, thus participating in AS (van Der Houven Van Oordt et al., 2000). ...
Alternative splicing (AS) is a critical means by which plants respond to changes in the environment, but few splicing factors contributing to AS have been reported and functionally characterized in rice (Oryza sativa L.). Here, we explored the function and molecular mechanism of the spliceosome‐associated protein OsFKBP20‐1b during AS.
We determined the AS landscape of wild‐type and osfkbp20‐1b knockout plants upon abscisic acid (ABA) treatment by transcriptome deep sequencing. To capture the dynamics of translating intron‐containing mRNAs, we blocked transcription with cordycepin and performed polysome profiling. We also analyzed whether OsFKBP20‐1b and the splicing factors OsSR34 and OsSR45 function together in AS using protoplast transfection assays.
We show that OsFKBP20‐1b interacts with OsSR34 and regulates its stability, suggesting a role as a chaperone‐like protein in the spliceosome. OsFKBP20‐1b facilitates the splicing of mRNAs with retained introns after ABA treatment; some of these mRNAs are translatable and encode functional transcriptional regulators of stress‐responsive genes. In addition, interacting proteins, OsSR34 and OsSR45, regulate the splicing of the same retained introns as OsFKBP20‐1b after ABA treatment.
Our findings reveal that spliceosome‐associated immunophilin functions in alternative RNA splicing in rice by positively regulating the splicing of retained introns to limit ABA response.
... Galectin-1 and Galectin-3 (Gal1 and Gal3) are components of U1 snRNP17 , 18 . Voss et al.19 propose a nuclear extract fractionation method to obtain Gal3-U1 snRNP-enriched complexes capable of rescuing splicing of U1 snRNP to 5' splice sites is an important mediator of splicing fidelity20 . Mutations at the 5' splice sites are critical and cause the loss of U1 snRNP pairing, resulting in splicing defects 2 . ...
... RBM39 bridges early spliceosome components and the pre-mRNA. RBM39, as well as its yeast homolog Rsd-1, were previously identified in early spliceosome complexes (Hegele et al., 2012;Shao et al., 2012). Thus, we investigated potential interactions between RBM39 and the spliceosome components using co-immunoprecipitation in HeLa cell nuclear extracts. ...
Pharmacologic depletion of RNA-binding motif 39 (RBM39) using aryl sulfonamides represents a promising anti-cancer therapy. However, its efficiency correlates with the expression level of DCAF15 which acts at the interface between RBM39, the drug and the E3-ubiquitin ligase. Consequently, the identification of alternative approaches to deplete RBM39 independently of DCAF15 is required. Here, we combined transcriptomic analysis, functional assays, and structural biology to elucidate the molecular mechanisms governing RBM39 homeostasis. Our data revealed that RBM39 autoregulates the splicing of its own pre-mRNA by triggering the inclusion of a poison exon. During this process, RBM39 selects the 3'-splice site of the toxic exon, helps the recruitment of U1 snRNP on its weak 5'-splice site and bridges the 3'-splice site recognition machinery. The elucidation of the molecular mechanisms controlling RBM39 homeostasis provides unprecedented insights into alternative 3'-splice site selection and a solid frame to design alternative anti-cancer therapies.
... An interaction between U1-70K and the 3 0ss-bound protein U2AF mediated by SR proteins has been reported in mammals (41). In fission yeast, the SR-like protein Rsd1 and Prp5 were reported to form a bridge between U1A and the U2 snRNP-specific protein SF3B1 (42,43). The human homolog of Rsd1, RBM39, was shown to bind SF3b155 (another component of U2 snRNP) and was found in prespliceosomal A complexes (40,44), suggesting alternative contacts that help bridging U1 and U2 snRNP in mammals. ...
Significance
Pre-messenger RNA (pre-mRNA) splicing is a key regulatory step in gene expression. The splicing reaction is mediated by the spliceosome, a dynamic complex comprising five small nuclear ribonucleoproteins (snRNPs), which assembles onto each intron in multiple steps. We present detailed structural analysis and supporting functional data of an important protein–RNA interaction between human U1 and U2 snRNP. Our structure shows that an intrinsically disordered arginine-glycine (RGG/RG)–rich motif of a U2 snRNP subunit forms an RNA-sequence–specific connection with U1 snRNP. This study broadens the functional scope of unstructured RGG/RG-rich motifs in RNA binding proteins and provides a molecular basis of early steps of spliceosome assembly, which may help develop innovative therapeutic strategies against diseases originating from splicing defects.
... This cross talk ultimately would help in the definition of the 5 0 ss and 3 0 ss of any single intron and would enable its splicing with single-nucleotide precision. An interaction between proteins at the 5 0 ss and 3 0 ss ends of introns was already described in fission yeast during a later step in splicing, where the helicase Prp11 assisted in doing the first ATP-dependent step in spliceosome assembly bridging U1 (through Rsd1) and U2 (through SF3b) (Shao et al., 2012). On the other hand, this mechanism is also partly reminiscent of the mechanism of transcription activation described in prokaryotes, where activation largely depends on the strength of the interaction between RNA polymerase and the promoter (Monsalve et al., 1997) and mutations at the promoter modulate the strength of this interaction. ...
Splicing of mRNA precursors is essential in the regulation of gene expression. U2AF65 recognizes the poly-pyrimidine tract and helps in the recognition of the branch point. Inactivation of fission yeast U2AF65 (Prp2) blocks splicing of most, but not all, pre-mRNAs, for reasons that are not understood. Here, we have determined genome-wide the splicing efficiency of fission yeast cells as they progress into synchronous meiosis in the presence or absence of functional Prp2. Our data indicate that in addition to the splicing elements at the 3′ end of any intron, the nucleotides immediately upstream the intron will determine whether Prp2 is required or dispensable for splicing. By changing those nucleotides in any given intron, we regulate its Prp2 dependency. Our results suggest a model in which Prp2 is required for the coordinated recognition of both intronic ends, placing Prp2 as a key regulatory element in the determination of the exon-intron boundaries.
... However, about 40 of the genes had increased or decreased expression levels in conidia, suggesting that differential expression of some components of the spliceosome may be important for the alternative splicing. For example, SF3b1 (MGG_00356), which is essential for intron definition in yeast (Shao et al., 2012), was much more expressed in conidia than in hyphae (Supplementary Figure 5A). We also compared the expression levels of genes essential for nonsense-mediated decay (NMD; Chang et al., 2007;Kanehisa et al., 2012), and observed that UPF1 (MGG_03004) was much more expressed in hyphae than in conidia (Supplementary Figure 5B) although almost all the other NMD-related genes had similar expression levels in conidia and hyphae (Figure 8). ...
Pyricularia oryzae causes the rice blast, which is one of the most devastating crop diseases worldwide, and is a model fungal pathogen widely used for dissecting the molecular mechanisms underlying fungal virulence/pathogenicity. Although the whole genome sequence of P. oryzae is publicly available, its current transcriptomes remain incomplete, lacking the information on non-protein coding genes and alternative splicing. Here, we performed and analyzed RNA-Seq of conidia and hyphae, resulting in the identification of 3,374 novel genes. Interestingly, the vast majority of these novel genes likely transcribed long non-coding RNAs (lncRNAs), and most of them were localized in the intergenic regions. Notably, their expressions were concomitant with the transcription of neighboring genes thereof in conidia and hyphae. In addition, 2,358 genes were found to undergo alternative splicing events. Furthermore, we exemplified that a lncRNA was important for hyphal growth likely by regulating the neighboring protein-coding gene and that alternative splicing of the transcription factor gene CON7 was required for appressorium formation. In summary, results from this study indicate that lncRNA transcripts and alternative splicing events are two important mechanisms for regulating the expression of genes important for conidiation, hyphal growth, and pathogenesis, and provide new insights into transcriptomes and gene regulation in the rice blast fungus.
... Human and fission yeast Prp5 have been shown to interact with U1 and U2 through distinct domains (22). SpPrp5 directly binds U2 component SF3b, and also interacts with U1 through SR protein Rsd1 in bridging U1 and U2 (45). Thus, U2 can be recruited to the CC by interacting with U1, and upon binding to CC2, is presented with a pre-mRNA region downstream of the Msl5-Mud2 binding site. ...
Splicing of pre-mRNA is initiated by binding of U1 to the 5′ splice site and of Msl5-Mud2 heterodimer to the branch site (BS). Subsequent binding of U2 displaces Msl5-Mud2 from the BS to form the prespliceosome, a step governing branchpoint selection and hence 3′ splice site choice, and linking splicing to myelodysplasia and many cancers in human. Two DEAD-box proteins, Prp5 and Sub2, are required for this step, but neither is stably associated with the pre-mRNA during the reaction. Using BS-mutated ACT1 pre-mRNA, we previously identified a splicing intermediate complex, FIC, which contains U2 and Prp5, but cannot bind the tri-snRNP. We show here that Msl5 remains associated with the upstream cryptic branch site (CBS) in the FIC, with U2 binding a few bases downstream of the BS. U2 mutants that restore U2-BS base pairing enable dissociation of Prp5 and allows splicing to proceed. The CBS is required for splicing rescue by compensatory U2 mutants, and for formation of FIC, demonstrating a role for Msl5 in directing U2 to the BS, and of U2-BS base pairing for release of Prp5 and Msl5-Mud2 to form the prespliceosome. Our results provide insights into how the prespliceosome may form in normal splicing reaction.
