scholarly journals Assembly of the U5 snRNP component PRPF8 is controlled by the HSP90/R2TP chaperones

2017 ◽  
Vol 216 (6) ◽  
pp. 1579-1596 ◽  
Author(s):  
Anna Malinová ◽  
Zuzana Cvačková ◽  
Daniel Matějů ◽  
Zuzana Hořejší ◽  
Claire Abéza ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Dependent Manner ◽  
Proteomics Data ◽  
Small Nuclear Ribonucleoprotein ◽  
Crucial Component ◽  
Potential Link ◽  
Nuclear Ribonucleoprotein ◽  
New Interactions

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.

scholarly journals The pre-mRNA-splicing factor SF3a66 functions as a microtubule-binding and -bundling protein

2004 ◽  
Vol 382 (1) ◽  
pp. 223-230 ◽  
Author(s):  
Kei TAKENAKA ◽  
Hiroyuki NAKAGAWA ◽  
Shigeaki MIYAMOTO ◽  
Hiroaki MIKI
Keyword(s):  
Rna Splicing ◽  
Ectopic Expression ◽  
Neuroblastoma Cells ◽  
High Molecular Mass ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Canonical Function ◽  
High Affinity ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

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.

scholarly journals PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle

1989 ◽  
Vol 9 (9) ◽  
pp. 3710-3719
Author(s):  
J Banroques ◽  
J N Abelson
Keyword(s):  
Essential Role ◽  
Mrna Splicing ◽  
Ribonucleoprotein Particle ◽  
Specific Antibodies ◽  
Small Nuclear Ribonucleoprotein ◽  
Assembly Pathway ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

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.

scholarly journals Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins.

1995 ◽  
Vol 15 (1) ◽  
pp. 445-455 ◽  
Author(s):  
J Roy ◽  
B Zheng ◽  
B C Rymond ◽  
J L Woolford
Keyword(s):  
Protein Complexes ◽  
Mrna Splicing ◽  
Yeast Cells ◽  
Ribonucleoprotein Particle ◽  
Sm Proteins ◽  
Small Nuclear Ribonucleoprotein ◽  
Snrnp Protein ◽  
Nuclear Ribonucleoprotein ◽  
Precursor Mrna

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.

scholarly journals Human Cancer-Associated Mutations of SF3B1 Lead to a Splicing Modification of Its Own RNA

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 652
Author(s):  
Tiffany Bergot ◽  
Eric Lippert ◽  
Nathalie Douet-Guilbert ◽  
Séverine Commet ◽  
Laurent Corcos ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Cancer ◽  
Myeloid Cell ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Small Nuclear Ribonucleoprotein ◽  
Genes Encoding ◽  
Protein Isoform ◽  
Nuclear Ribonucleoprotein

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.

scholarly journals Novel Splicing Factor RBM25 Modulates Bcl-x Pre-mRNA 5′ Splice Site Selection

2008 ◽  
Vol 28 (19) ◽  
pp. 5924-5936 ◽  
Author(s):  
AnYu Zhou ◽  
Alexander C. Ou ◽  
Aeri Cho ◽  
Edward J. Benz ◽  
Shu-Ching Huang
Keyword(s):  
Splice Site ◽  
Dependent Manner ◽  
Splice Site Selection ◽  
Exon 2 ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp ◽  
Nuclear Ribonucleoprotein ◽  
Dose Dependent ◽  
Selection Of

ABSTRACT RBM25 has been shown to associate with splicing cofactors SRm160/300 and assembled splicing complexes, but little is known about its splicing regulation. Here, we characterize the functional role of RBM25 in alternative pre-mRNA splicing. Increased RBM25 expression correlated with increased apoptosis and specifically affected the expression of Bcl-x isoforms. RBM25 stimulated proapoptotic Bcl-xS 5′ splice site (5′ ss) selection in a dose-dependent manner, whereas its depletion caused the accumulation of antiapoptotic Bcl-xL. Furthermore, RBM25 specifically bound to Bcl-x RNA through a CGGGCA sequence located within exon 2. Mutation in this element abolished the ability of RBM25 to enhance Bcl-xS 5′ ss selection, leading to decreased Bcl-xS isoform expression. Binding of RBM25 was shown to promote the recruitment of the U1 small nuclear ribonucleoprotein particle (snRNP) to the weak 5′ ss; however, it was not required when a strong consensus 5′ ss was present. In support of a role for RBM25 in modulating the selection of a 5′ ss, we demonstrated that RBM25 associated selectively with the human homolog of yeast U1 snRNP-associated factor hLuc7A. These data suggest a novel mode for Bcl-xS 5′ ss activation in which binding of RBM25 with exonic element CGGGCA may stabilize the pre-mRNA-U1 snRNP through interactions with hLuc7A.

scholarly journals SPF45-related splicing factor for phytochrome signaling promotes photomorphogenesis by regulating pre-mRNA splicing in Arabidopsis

2017 ◽  
Vol 114 (33) ◽  
pp. E7018-E7027 ◽  
Author(s):  
Ruijiao Xin ◽  
Ling Zhu ◽  
Patrice A. Salomé ◽  
Estefania Mancini ◽  
Carine M. Marshall ◽  
...  
Keyword(s):  
Gene Expression ◽  
Circadian Clock ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Clock Genes ◽  
Mrna Processing ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Light Signaling ◽  
Small Nuclear Ribonucleoprotein

Light signals regulate plant growth and development by controlling a plethora of gene expression changes. Posttranscriptional regulation, especially pre-mRNA processing, is a key modulator of gene expression; however, the molecular mechanisms linking pre-mRNA processing and light signaling are not well understood. Here we report a protein related to the human splicing factor 45 (SPF45) named splicing factor for phytochrome signaling (SFPS), which directly interacts with the photoreceptor phytochrome B (phyB). In response to light, SFPS-RFP (red fluorescent protein) colocalizes with phyB-GFP in photobodies. sfps loss-of-function plants are hyposensitive to red, far-red, and blue light, and flower precociously. SFPS colocalizes with U2 small nuclear ribonucleoprotein-associated factors including U2AF65B, U2A′, and U2AF35A in nuclear speckles, suggesting SFPS might be involved in the 3′ splice site determination. SFPS regulates pre-mRNA splicing of a large number of genes, of which many are involved in regulating light signaling, photosynthesis, and the circadian clock under both dark and light conditions. In vivo RNA immunoprecipitation (RIP) assays revealed that SFPS associates with EARLY FLOWERING 3 (ELF3) mRNA, a critical link between light signaling and the circadian clock. Moreover, PHYTOCHROME INTERACTING FACTORS (PIFs) transcription factor genes act downstream of SFPS, as the quadruple pif mutant pifq suppresses defects of sfps mutants. Taken together, these data strongly suggest SFPS modulates light-regulated developmental processes by controlling pre-mRNA splicing of light signaling and circadian clock genes.

scholarly journals A U5 small nuclear ribonucleoprotein particle protein involved only in the second step of pre-mRNA splicing in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (5) ◽  
pp. 2959-2970
Author(s):  
D S Horowitz ◽  
J Abelson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Form ◽  
Mrna Splicing ◽  
Second Step ◽  
Temperature Sensitive ◽  
Ribonucleoprotein Particle ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

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.

scholarly journals A conserved epitope on a subset of SR proteins defines a larger family of Pre-mRNA splicing factors.

1995 ◽  
Vol 129 (4) ◽  
pp. 899-908 ◽  
Author(s):  
K M Neugebauer ◽  
J A Stolk ◽  
M B Roth
Keyword(s):  
Rna Polymerase Ii ◽  
Active Sites ◽  
Nuclear Extract ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Structural Element ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

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.

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