scholarly journals Association of nuclear matrix antigens with exon-containing splicing complexes.

1994 ◽  
Vol 127 (3) ◽  
pp. 593-607 ◽  
Author(s):  
B J Blencowe ◽  
J A Nickerson ◽  
R Issner ◽  
S Penman ◽  
P A Sharp
Keyword(s):  
Nuclear Matrix ◽  
Biological Data ◽  
Mrna Processing ◽  
Mrna Splicing ◽  
Nuclear Proteins ◽  
Second Step ◽  
Splicing Factors ◽  
Protein Splicing

mAbs raised against the human nuclear matrix (anti-NM)1 mAbs have been used to investigate the role of nuclear matrix antigens in pre-mRNA processing. The three anti-NM mAbs used in this study recognize antigens that are highly localized to nuclear matrix speckles. Surprisingly, all three of these mAbs preferentially immunoprecipitate splicing complexes containing exon sequences. The anti-NM mAbs efficiently immunoprecipitate the exon product complex but not complexes containing the lariat product after the second step of splicing. Two of the anti-NM mAbs completely inhibit pre-mRNA splicing in vitro. However, none of the anti-NM mAbs appear to recognize factors stably associated with splicing snRNPs. The three anti-NM mAbs predominantly react with distinct high molecular weight antigens, which belong to a class of nuclear proteins that selectively precipitate with Ser-Arg protein-splicing factors in the presence of high Mg2+ concentrations. Immunological, biochemical, and cell biological data indicate that two of the NM antigens are related to the defined set of Ser-Arg proteins. The results suggest the existence of an extended Ser-Arg family as a component of the nuclear matrix.

scholarly journals The WW Domain-Containing Proteins Interact with the Early Spliceosome and Participate in Pre-mRNA Splicing In Vivo

2004 ◽  
Vol 24 (20) ◽  
pp. 9176-9185 ◽  
Author(s):  
Kai-Ti Lin ◽  
Ruei-Min Lu ◽  
Woan-Yuh Tarn
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Ww Domain ◽  
Ww Domains ◽  
Small Nuclear Ribonucleoprotein

ABSTRACT A growing body of evidence supports the coordination of mRNA synthesis and its subsequent processing events. Nuclear proteins harboring both WW and FF protein interaction modules bind to splicing factors as well as RNA polymerase II and may serve to link transcription with splicing. To understand how WW domains coordinate the assembly of splicing complexes, we used glutathione S-transferase fusions containing WW domains from CA150 or FBP11 in pull-down experiments with HeLa cell nuclear extract. The WW domains associate preferentially with the U2 small nuclear ribonucleoprotein and with splicing factors SF1, U2AF, and components of the SF3 complex. Accordingly, WW domain-associating factors bind to the 3′ part of a pre-mRNA to form a pre-spliceosome-like complex. We performed both in vitro and in vivo splicing assays to explore the role of WW/FF domain-containing proteins in this process. However, although CA150 is associated with the spliceosome, it appears to be dispensable for splicing in vitro. Nevertheless, in vivo depletion of CA150 substantially reduced splicing efficiency of a reporter pre-mRNA. Moreover, overexpression of CA150 fragments containing both WW and FF domains activated splicing and modulated alternative exon selection, probably by facilitating 3′ splice site recognition. Our results suggest an essential role of WW/FF domain-containing factors in pre-mRNA splicing that likely occurs in concert with transcription in vivo.

scholarly journals Delta-secretase (AEP) mediates tau-splicing imbalance and accelerates cognitive decline in tauopathies

2018 ◽  
Vol 215 (12) ◽  
pp. 3038-3056 ◽  
Author(s):  
Zhi-Hao Wang ◽  
Pai Liu ◽  
Xia Liu ◽  
Shan Ping Yu ◽  
Jian-Zhi Wang ◽  
...  
Keyword(s):  
Cognitive Decline ◽  
Kinase Activity ◽  
Nuclear Translocation ◽  
Memory Deficits ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Synaptic Functions ◽  
Tau Isoforms ◽  
Exon 10

SRPK2 is abnormally activated in tauopathies including Alzheimer’s disease (AD). SRPK2 is known to play an important role in pre–mRNA splicing by phosphorylating SR-splicing factors. Dysregulation of tau exon 10 pre–mRNA splicing causes pathological imbalances in 3R- and 4R-tau, leading to neurodegeneration; however, the role of SRPK2 in these processes remains unclear. Here we show that delta-secretase (also known as asparagine endopeptidase; AEP), which is activated in AD, cleaves SRPK2 and increases its nuclear translocation as well as kinase activity, augmenting exon 10 inclusion. Conversely, AEP-uncleavable SRPK2 N342A mutant increases exon 10 exclusion. Lentiviral expression of truncated SRPK2 increases 4R-tau isoforms and accelerates cognitive decline in htau mice. Uncleavable SRPK2 N342A expression improves synaptic functions and prevents spatial memory deficits in tau intronic mutant FTDP-17 transgenic mice. Hence, AEP mediates tau-splicing imbalance in tauopathies via cleaving SRPK2.

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 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.

The splicing factor Prp17 interacts with the U2, U5 and U6 snRNPs and associates with the spliceosome pre- and post-catalysis

2008 ◽  
Vol 416 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Aparna K. Sapra ◽  
Piyush Khandelia ◽  
Usha Vijayraghavan
Keyword(s):  
Second Step ◽  
Splicing Factors ◽  
Temperature Sensitive ◽  
Biochemical Basis ◽  
Association Analyses ◽  
Small Nuclear Rnas ◽  
In Vitro Splicing ◽  
Mechanistic Basis ◽  
Small Nuclear Ribonucleoproteins

Saccharomyces cerevisiae PRP17-null mutants are temperature-sensitive for growth. In vitro splicing with extracts lacking Prp17 are kinetically slow for the first step of splicing and are arrested for the second step at temperatures greater than 34 °C. In the present study we show that these stalled spliceosomes are compromised for an essential conformational switch that is triggered by Prp16 helicase. These results suggest a plausible mechanistic basis for the second-step arrest in prp17Δ extracts and support a role for Prp17 in conjunction with Prp16. To understand the association of Prp17 with spliceosomes we used a functional epitope-tagged protein in co-immunoprecipitation experiments. Examination of co-precipitated snRNAs (small nuclear RNAs) show that Prp17 interacts with U2, U5 and U6 snRNPs (small nuclear ribonucleoproteins) but it is not a core component of any one snRNP. Prp17 association with in-vitro-assembled spliceosome complexes on actin pre-mRNAs was also investigated. Although the U5 snRNP proteins Prp8 and Snu114 are found in early pre-spliceosomes that contain all five snRNPs, Prp17 is not detectable at this step; however, Prp17 is present in the subsequent pre-catalytic A1 complex, containing unspliced pre-mRNA, formed after the dissociation of U4 snRNP. Thus Prp17 joins the spliceosome prior to both catalytic reactions. Our results indicate continued interactions in catalytic spliceosomes that contain reaction intermediates and in post-splicing complexes containing the lariat intron. These Prp17–spliceosome association analyses provide a biochemical basis for the delayed first step in prp17Δ and explain the previously known multiple genetic interactions between Prp17, factors of the Prp19-complex [NTC (nineteen complex)], functional elements in U2 and U5 snRNAs and other second-step splicing factors.

scholarly journals Rapid-Response Splicing Reporter Screens Identify Differential Regulators of Constitutive and Alternative Splicing

2010 ◽  
Vol 30 (7) ◽  
pp. 1718-1728 ◽  
Author(s):  
Ihab Younis ◽  
Michael Berg ◽  
Daisuke Kaida ◽  
Kimberly Dittmar ◽  
Congli Wang ◽  
...  
Keyword(s):  
Differential Regulation ◽  
Selective Inhibitor ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Protein Splicing ◽  
Reporter System ◽  
Potential Toxicity ◽  
Sr Protein ◽  
Cellular Processes ◽  
Pharmacologically Active

ABSTRACT Bioactive compounds have been invaluable for dissecting the mechanisms, regulation, and functions of cellular processes. However, very few such reagents have been described for pre-mRNA splicing. To facilitate their systematic discovery, we developed a high-throughput cell-based assay that measures pre-mRNA splicing by utilizing a quantitative reporter system with advantageous features. The reporter, consisting of a destabilized, intron-containing luciferase expressed from a short-lived mRNA, allows rapid screens (<4 h), thereby obviating the potential toxicity of splicing inhibitors. We describe three inhibitors (out of >23,000 screened), all pharmacologically active: clotrimazole, flunarizine, and chlorhexidine. Interestingly, none was a general splicing inhibitor. Rather, each caused distinct splicing changes of numerous genes. We further discovered the target of action of chlorhexidine and show that it is a selective inhibitor of specific Cdc2-like kinases (Clks) that phosphorylate serine-arginine-rich (SR) protein splicing factors. Our findings reveal unexpected activities of clinically used drugs in splicing and uncover differential regulation of constitutively spliced introns.

scholarly journals Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.

1989 ◽  
Vol 9 (12) ◽  
pp. 5273-5280 ◽  
Author(s):  
G C Conway ◽  
A R Krainer ◽  
D L Spector ◽  
R J Roberts
Keyword(s):  
Rna Splicing ◽  
De Novo ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Macromolecular Complex ◽  
Large Structures ◽  
Nuclear Extracts ◽  
In Vitro Splicing ◽  
Nuclear Ribonucleoprotein

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.

scholarly journals Nuclear organisation of NIPP1, a regulatory subunit of protein phosphatase 1 that associates with pre-mRNA splicing factors

1999 ◽  
Vol 112 (2) ◽  
pp. 157-168 ◽  
Author(s):  
L. Trinkle-Mulcahy ◽  
P. Ajuh ◽  
A. Prescott ◽  
F. Claverie-Martin ◽  
S. Cohen ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Mrna Splicing ◽  
Dominant Negative ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Dominant Negative Mutant ◽  
Splicing Factors ◽  
Nuclear Extracts ◽  
Nuclear Organisation

Protein phosphatase-1 (PP1) is complexed to many proteins that target it to particular subcellular locations and regulate its activity. Here, we show that ‘nuclear inhibitor of PP1’ (NIPP1), a major nuclear PP1-binding protein, shows a speckled nucleoplasmic distribution where it is colocalised with pre-mRNA splicing factors. One of these factors (Sm) is also shown to be complexed to NIPP1 in nuclear extracts. Immunodepletion of NIPP1 from nuclear extracts, or addition of a ‘dominant negative’ mutant lacking a functional PP1 binding site, greatly reduces pre-mRNA splicing activity in vitro. These findings implicate the NIPP1-PP1 complex in the control of pre-mRNA splicing.

scholarly journals Regulation of neuronal mRNA splicing and Tau isoform ratio by ATXN3 through deubiquitylation of splicing factors

2019 ◽  
Author(s):  
Andreia Neves-Carvalho ◽  
Sara Duarte-Silva ◽  
Joana Silva ◽  
Bruno Almeida ◽  
Sasja Heetveld ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
Rna Metabolism ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Loss Of Function ◽  
Relevant Role ◽  
The Brain ◽  
Precise Role ◽  
Exon 10

ABSTRACTThe ubiquitylation/deubiquitylation balance in cells is maintained by Deubiquitylating enzymes, including ATXN3. The precise role of this protein, mutated in SCA3, remains elusive, as few substrates for its deubiquitylating activity were identified. Therefore, we characterized the ubiquitome of neuronal cells lacking ATXN3, and found altered polyubiquitylation in a large proportion of proteins involved in RNA metabolism, including splicing factors. Using transcriptomic analysis and reporter minigenes we confirmed that splicing was globally altered in these cells. Among the targets with altered splicing was SRSF7 (9G8), a key regulator of MAPT (Tau) exon 10 splicing. Loss-of-function of ATXN3 led to a deregulation of MAPT exon 10 splicing resulting in a decreased 4R/3R-Tau ratio. Similar alterations were found in the brain of a SCA3 mouse and humans, pointing to a relevant role of this mechanism in SCA3, and establishing a previously unsuspected link between two key proteins involved in different neurodegenerative disorders.

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