scholarly journals A UHM - ULM interface contributes to U2AF2 and SF3B1 association for pre-mRNA splicing

2022 ◽  
Author(s):  
Justin Galardi ◽  
Victoria N Bela ◽  
Nazish Jeffery ◽  
Xueyang He ◽  
Eliezra Glasser ◽  
...  

In the early stages of spliceosome assembly, the 3' splice site is recognized by sequential complexes of U2AF2 with SF1 followed by the SF3B1 subunit of the U2 small nuclear ribonucleoprotein particle. The U2AF2 - SF1 interface comprises a U2AF homology motif (UHM) of U2AF2 and a well-characterized U2AF ligand motif (ULM)/coiled coil region of SF1. However, the structure of the U2AF2 - SF3B1 interface and its importance for pre-mRNA splicing is unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8 Å resolution. The trajectory of the SF3B1 ULM across the U2AF2 UHM surface differed from prior UHM/ULM structures. This distinctive structure is expected to modulate the orientations of the full-length proteins. Using isothermal titration calorimetry, we established similar binding affinities of a minimal U2AF2 UHM - SF3B1 ULM complex and a nearly full-length U2AF2 protein binding the N-terminal SF3B1 region, with or without an auxiliary SF3B6 subunit. We showed that key residues at the U2AF2 UHM - SF3B1 ULM interface are required for high affinity association and co-immunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2 - SF3B1 interface altered splicing of representative human transcripts. Further analysis of these transcripts and genome-wide data sets indicated that the subset of splice sites co-regulated by U2AF2 and SF3B1 are largely distinct from those co-regulated by U2AF2 and SF1. Altogether, these findings support distinct structural and functional roles for the sequential SF1 and SF3B1 complexes with U2AF2 during the pre-mRNA splicing process.

1989 ◽  
Vol 9 (9) ◽  
pp. 3710-3719
Author(s):  
J Banroques ◽  
J N Abelson

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex.


1995 ◽  
Vol 15 (1) ◽  
pp. 445-455 ◽  
Author(s):  
J Roy ◽  
B Zheng ◽  
B C Rymond ◽  
J L Woolford

Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins. The Sm proteins also play a pivotal role in metazoan snRNP biogenesis. We have characterized a Saccharomyces cerevisiae gene, SMD3, that encodes the core snRNP protein Smd3. The Smd3 protein is required for pre-mRNA splicing in vivo. Depletion of this protein from yeast cells affects the levels of U snRNAs and their cap modification, indicating that Smd3 is required for snRNP biogenesis. Smd3 is structurally and functionally distinct from the previously described yeast core polypeptide Smd1. Although Smd3 and Smd1 are both associated with the spliceosomal snRNPs, overexpression of one cannot compensate for the loss of the other. Thus, these two proteins have distinct functions. A pool of Smd3 exists in the yeast cytoplasm. This is consistent with the possibility that snRNP assembly in S. cerevisiae, as in metazoans, is initiated in the cytoplasm from a pool of RNA-free core snRNP protein complexes.


Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 652
Author(s):  
Tiffany Bergot ◽  
Eric Lippert ◽  
Nathalie Douet-Guilbert ◽  
Séverine Commet ◽  
Laurent Corcos ◽  
...  

Deregulation of pre-mRNA splicing is observed in many cancers and hematological malignancies. Genes encoding splicing factors are frequently mutated in myelodysplastic syndromes, in which SF3B1 mutations are the most frequent. SF3B1 is an essential component of the U2 small nuclear ribonucleoprotein particle that interacts with branch point sequences close to the 3’ splice site during pre-mRNA splicing. SF3B1 mutations mostly lead to substitutions at restricted sites in the highly conserved HEAT domain, causing a modification of its function. We found that SF3B1 was aberrantly spliced in various neoplasms carrying an SF3B1 mutation, by exploring publicly available RNA sequencing raw data. We aimed to characterize this novel SF3B1 transcript, which is expected to encode a protein with an insertion of eight amino acids in the H3 repeat of the HEAT domain. We investigated the splicing proficiency of this SF3B1 protein isoform, in association with the most frequent mutation (K700E), through functional complementation assays in two myeloid cell lines stably expressing distinct SF3B1 variants. The yeast Schizosaccharomyces pombe was also used as an alternative model. Insertion of these eight amino acids in wild-type or mutant SF3B1 (K700E) abolished SF3B1 essential function, highlighting the crucial role of the H3 repeat in the splicing function of SF3B1.


2017 ◽  
Vol 216 (6) ◽  
pp. 1579-1596 ◽  
Author(s):  
Anna Malinová ◽  
Zuzana Cvačková ◽  
Daniel Matějů ◽  
Zuzana Hořejší ◽  
Claire Abéza ◽  
...  

Splicing is catalyzed by the spliceosome, a complex of five major small nuclear ribonucleoprotein particles (snRNPs). The pre-mRNA splicing factor PRPF8 is a crucial component of the U5 snRNP, and together with EFTUD2 and SNRNP200, it forms a central module of the spliceosome. Using quantitative proteomics, we identified assembly intermediates containing PRPF8, EFTUD2, and SNRNP200 in association with the HSP90/R2TP complex, its ZNHIT2 cofactor, and additional proteins. HSP90 and R2TP bind unassembled U5 proteins in the cytoplasm, stabilize them, and promote the formation of the U5 snRNP. We further found that PRPF8 mutants causing Retinitis pigmentosa assemble less efficiently with the U5 snRNP and bind more strongly to R2TP, with one mutant retained in the cytoplasm in an R2TP-dependent manner. We propose that the HSP90/R2TP chaperone system promotes the assembly of a key module of U5 snRNP while assuring the quality control of PRPF8. The proteomics data further reveal new interactions between R2TP and the tuberous sclerosis complex (TSC), pointing to a potential link between growth signals and the assembly of key cellular machines.


2004 ◽  
Vol 382 (1) ◽  
pp. 223-230 ◽  
Author(s):  
Kei TAKENAKA ◽  
Hiroyuki NAKAGAWA ◽  
Shigeaki MIYAMOTO ◽  
Hiroaki MIKI

SF3a (splicing factor 3a) complex is an essential component of U2 snRNPs (small nuclear ribonucleoprotein particles), which are involved in pre-mRNA splicing. This complex consists of three subunits: SF3a60, SF3a66 and SF3a120. Here, we report a possible non-canonical function of a well-characterized RNA-splicing factor, SF3a66. Ectopic expression experiments using each SF3a subunit in N1E 115 neuroblastoma cells reveals that SF3a66 alone can induce neurite extension, suggesting that SF3a66 functions in the regulation of cell morphology. A screen for proteins that bind to SF3a66 clarifies that SF3a66 binds to β-tubulin, and also to microtubules, with high affinity, indicating that SF3a66 is a novel MAP (microtubule-associated protein). Electron microscopy experiments show that SF3a66 can bundle microtubules, and that bundling of microtubules is due to cross-bridging of microtubules by high-molecular-mass complexes of oligomerized SF3a66. These results indicate that SF3a66 is likely to be a novel MAP, and can function as a microtubule-bundling protein independently of RNA splicing.


1993 ◽  
Vol 13 (5) ◽  
pp. 2959-2970
Author(s):  
D S Horowitz ◽  
J Abelson

The PRP18 gene, which had been identified in a screen for pre-mRNA splicing mutants in Saccharomyces cerevisiae, has been cloned and sequenced. Yeast strains bearing only a disrupted copy of PRP18 are temperature sensitive for growth; even at a low temperature, they grow extremely slowly and do not splice pre-mRNA efficiently. This unusual temperature sensitivity can be reproduced in vitro; extracts immunodepleted of PRP18 are temperature sensitive for the second step of splicing. The PRP18 protein has been overexpressed in active form in Escherichia coli and has been purified to near homogeneity. Antibodies directed against PRP18 precipitate the U4/U5/U6 small nuclear ribonucleoprotein particle (snRNP) from yeast extracts. From extracts depleted of the U6 small nuclear RNA (snRNA), the U4 and U5 snRNAs can be immunoprecipitated, while no snRNAs can be precipitated from extracts depleted of the U5 snRNA. PRP18 therefore appears to be primarily associated with the U5 snRNP. The antibodies against PRP18 inhibit the second step of pre-mRNA splicing in vitro. Together, these results imply that the U5 snRNP plays a role in the second step of splicing and suggest a model for the action of PRP18.


1995 ◽  
Vol 129 (4) ◽  
pp. 899-908 ◽  
Author(s):  
K M Neugebauer ◽  
J A Stolk ◽  
M B Roth

The removal of introns from eukaryotic pre-mRNA occurs in a large ribonucleoprotein complex called the spliceosome. We have generated a monoclonal antibody (mAb 16H3) against four of the family of six SR proteins, known regulators of splice site selection and spliceosome assembly. In addition to the reactive SR proteins, SRp20, SRp40, SRp55, and SRp75, mAb 16H3 also binds approximately 20 distinct nuclear proteins in human, frog, and Drosophila extracts, whereas yeast do not detectably express the epitope. The antigens are shown to be nuclear, nonnucleolar, and concentrated at active sites of RNA polymerase II transcription which suggests their involvement in pre-mRNA processing. Indeed, most of the reactive proteins observed in nuclear extract are detected in spliceosomes (E and/or B complex) assembled in vitro, including the U1 70K component of the U1 small nuclear ribonucleoprotein particle and both subunits of U2AF. Interestingly, the 16H3 epitope was mapped to a 40-amino acid polypeptide composed almost exclusively of arginine alternating with glutamate and aspartate. All of the identified antigens, including the human homolog of yeast Prp22 (HRH1), contain a similar structural element characterized by arginine alternating with serine, glutamate, and/or aspartate. These results indicate that many more spliceosomal components contain such arginine-rich domains. Because it is conserved among metazoans, we propose that the "alternating arginine" domain recognized by mAb 16H3 may represent a common functional element of pre-mRNA splicing factors.


2015 ◽  
Vol 112 (11) ◽  
pp. E1191-E1200 ◽  
Author(s):  
Martha R. Stark ◽  
Elizabeth A. Dunn ◽  
William S. C. Dunn ◽  
Cameron J. Grisdale ◽  
Anthony R. Daniele ◽  
...  

The human spliceosome is a large ribonucleoprotein complex that catalyzes pre-mRNA splicing. It consists of five snRNAs and more than 200 proteins. Because of this complexity, much work has focused on the Saccharomyces cerevisiae spliceosome, viewed as a highly simplified system with fewer than half as many splicing factors as humans. Nevertheless, it has been difficult to ascribe a mechanistic function to individual splicing factors or even to discern which are critical for catalyzing the splicing reaction. We have identified and characterized the splicing machinery from the red alga Cyanidioschyzon merolae, which has been reported to harbor only 26 intron-containing genes. The U2, U4, U5, and U6 snRNAs contain expected conserved sequences and have the ability to adopt secondary structures and form intermolecular base-pairing interactions, as in other organisms. C. merolae has a highly reduced set of 43 identifiable core splicing proteins, compared with ∼90 in budding yeast and ∼140 in humans. Strikingly, we have been unable to find a U1 snRNA candidate or any predicted U1-associated proteins, suggesting that splicing in C. merolae may occur without the U1 small nuclear ribonucleoprotein particle. In addition, based on mapping the identified proteins onto the known splicing cycle, we propose that there is far less compositional variability during splicing in C. merolae than in other organisms. The observed reduction in splicing factors is consistent with the elimination of spliceosomal components that play a peripheral or modulatory role in splicing, presumably retaining those with a more central role in organization and catalysis.


2016 ◽  
Vol 113 (19) ◽  
pp. 5447-5452 ◽  
Author(s):  
Xian Deng ◽  
Tiancong Lu ◽  
Lulu Wang ◽  
Lianfeng Gu ◽  
Jing Sun ◽  
...  

Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is involved in a multitude of biological processes in eukaryotes. Symmetric arginine dimethylation mediated by PRMT5 modulates constitutive and alternative pre-mRNA splicing of diverse genes to regulate normal growth and development in multiple species; however, the underlying molecular mechanism remains largely unknown. A genetic screen for suppressors of an Arabidopsis symmetric arginine dimethyltransferase mutant, atprmt5, identified two gain-of-function alleles of pre-mRNA processing factor 8 gene (prp8-8 and prp8-9), the highly conserved core component of the U5 small nuclear ribonucleoprotein (snRNP) and the spliceosome. These two atprmt5 prp8 double mutants showed suppression of the developmental and splicing alterations of atprmt5 mutants. In atprmt5 mutants, the NineTeen complex failed to be assembled into the U5 snRNP to form an activated spliceosome; this phenotype was restored in the atprmt5 prp8-8 double mutants. We also found that loss of symmetric arginine dimethylation of Sm proteins prevents recruitment of the NineTeen complex and initiation of spliceosome activation. Together, our findings demonstrate that symmetric arginine dimethylation has important functions in spliceosome assembly and activation, and uncover a key molecular mechanism for arginine methylation in pre-mRNA splicing that impacts diverse developmental processes.


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