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 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 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.

scholarly journals In vitro splicing of pre-messenger RNA with extracts from 5-fluorouridine-treated cells

1994 ◽  
Vol 297 (2) ◽  
pp. 297-301 ◽  
Author(s):  
J R Patton
Keyword(s):  
Cell Growth ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Significant Inhibition ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
Specific Step ◽  
In Vitro Splicing ◽  
Nuclear Ribonucleoprotein

The effect of 5-fluorouridine (5-FU) treatment of cells on the splicing of pre-mRNA was determined using cellular extracts and splicing in vitro. Nuclear extracts from control cells and cells treated with 5-FU were prepared and used to splice pre-mRNAs in vitro. The drug treatment resulted in inhibition of cell growth but had little effect on RNA synthesis. The extracts from 5-FU-treated cells showed significant inhibition of splicing. This inhibition was the result of reduced efficiency and was not caused by a block at a specific step in the splicing pathway. There were no observable changes in the levels or physical properties of the small nuclear ribonucleoprotein particles that are essential cofactors in the splicing process. The deficiency in splicing in the extracts from 5-FU-treated cells could be supplemented by the addition of complementary fractions from a control extract.

Structure of spliceosomal UsnRNPs

1992 ◽  
Vol 50 (1) ◽  
pp. 460-461
Author(s):  
Reinhard Lührmann ◽  
Sven-Erik Behrens ◽  
Berthold Kastner
Keyword(s):  
Immunoaffinity Chromatography ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Nuclear Extracts ◽  
U2 Snrnp ◽  
Specific Proteins ◽  
The Common ◽  
The Individual ◽  
Stable Formation

The major snRNPs, Ul, U2, U4/U6 and U5, are essential trans-acting factors in the pre-mRNA splicing process. They assemble with a pre-mRNA and a number of other non-snRNP splicing factors prior to the splicing reaction to form an active spliceosome. We are interested in investigating the biochemical composition of UsnRNPs and their ultrastructure as well as their function in splicing. In HeLa cell nuclear extracts the spliceosomal UsnRNPs exhibit differential association behaviour depending on the salt concentration. Thus, at high salt (420 mM) the majority of the Ul, U2, U4/U6 snRNPs migrates on sucrose gradients at 10-12S, while U5 snRNP sediments at 20S. Under in vitro splicing conditions (i.e. at about 100 mM salt), U5 and U4/U6 snRNPs form a 25 S [U4/U6.U5]tri-snRNP-complex and U2 snRNPs sediment at about 17 S.We have isolated the various types of UsnRNPs under native conditions using mainly immunoaffinity chromatography procedures. Today we can distinguish more than 35 distinct snRNP proteins. They can be grouped into two classes. The first class comprises eight common snRNP proteins which are present in each of the spliceosomal UsnRNPs. In addition, the individual snRNPs contain snRNP-specific proteins. These include three (70k, A, C) for the 12 S Ul snRNP, two (A′, B″ for the 12 S U2 snRNP, an additional eight for the 17 S U2 snRNP and eight for the 20 S U5 snRNP. The 25 S [U4/U6.U5]tri-snRNP-complex contains, in addition to the common proteins and the U5-specific proteins, a third group of six proteins which are essential for the stable formation of the tri-snRNP-complex. Thus, the different S-values of a particular snRNP particle result from differences in the population of snRNP-specific proteins associated with that particle.

scholarly journals Participation of the C-Terminal Domain of RNA Polymerase II in Exon Definition during Pre-mRNA Splicing

2000 ◽  
Vol 20 (21) ◽  
pp. 8290-8301 ◽  
Author(s):  
Changqing Zeng ◽  
Susan M. Berget
Keyword(s):  
Rna Polymerase Ii ◽  
Repeat Sequence ◽  
Splicing Factors ◽  
Exon Definition ◽  
Pol Ii ◽  
Terminal Domain ◽  
Nuclear Extracts ◽  
In Vitro Splicing ◽  
Precursor Rna

ABSTRACT Interaction between transcription and pre-mRNA processing via binding of polymerase II (Pol II) to factors involved in capping, splicing, and polyadenylation has recently been demonstrated. The C-terminal domain (CTD), a highly phosphorylated repeat sequence of the largest subunit of Pol II, has been implicated in this interaction because deletion of this domain affects downstream RNA processing events and because it is the binding site for numerous processing factors. Here we show that recombinant CTD, free of other components of Pol II, activated in vitro splicing and assembly of the spliceosome in nuclear extracts if, and only if, the assayed precursor RNA was recognized via exon definition, i.e., if the substrates contained complete exons with both 3′ and 5′ splice sites. Furthermore, depletion of intact Pol II inactivated splicing of this set of precursor RNAs and addition of recombinant CTD restored activity. The added recombinant CTD was quickly hyper- and hypophosphorylated in extract, became associated with the precursor RNA, and stimulated the association of U1 snRNPs but not ASF/SF2 with substrate RNA. These observations suggest that the mode of interaction between the CTD and splicing factors is integrally tied to exon definition and the mechanism whereby distal exons can be recognized and brought into juxtaposition during assembly of the spliceosome.

Functional association of nuclear protein 4.1 with pre-mRNA splicing factors

1998 ◽  
Vol 111 (14) ◽  
pp. 1963-1971 ◽  
Author(s):  
M.J. Lallena ◽  
C. Martinez ◽  
J. Valcarcel ◽  
I. Correas
Keyword(s):  
Nuclear Protein ◽  
Transmembrane Proteins ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Protein 4.1 ◽  
Nuclear Extracts ◽  
Nuclear Structures ◽  
The Relationship

Protein 4.1 is a multifunctional polypeptide that links transmembrane proteins with the underlying spectrin/actin cytoskeleton. Recent studies have shown that protein 4.1 is also present in the nucleus, localized in domains enriched in splicing factors. Here we further analyze the relationship between protein 4. 1 and components of the splicing machinery. Using HeLa nuclear extracts capable of supporting the splicing of pre-mRNAs in vitro, we show that anti-4.1 antibodies specifically immunoprecipitate pre-mRNA and splicing intermediates. Immunodepletion of protein 4.1 from HeLa nuclear extracts results in inhibition of their splicing activity, as assayed with two different pre-mRNA substrates. Coprecipitation of protein 4.1 from HeLa nuclear extracts with proteins involved in the processing of pre-mRNA further suggests an association between nuclear protein 4.1 and components of the splicing apparatus. The molecular cloning of a 4.1 cDNA encoding the isoform designated 4.1E has allowed us to show that this protein is targeted to the nucleus, that it associates with the splicing factor U2AF35, and that its overexpression induces the redistribution of the splicing factor SC35. Based on our combined biochemical and localization results, we propose that 4.1 proteins are part of nuclear structures to which splicing factors functionally associate, most likely for storage purposes.

scholarly journals Conserved Loop I of U5 Small Nuclear RNA Is Dispensable for Both Catalytic Steps of Pre-mRNA Splicing in HeLa Nuclear Extracts

1999 ◽  
Vol 19 (4) ◽  
pp. 2782-2790 ◽  
Author(s):  
Véronique Ségault ◽  
Cindy L. Will ◽  
Maria Polycarpou-Schwarz ◽  
Iain W. Mattaj ◽  
Christiane Branlant ◽  
...  
Keyword(s):  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Internal Loop ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein ◽  
U5 Snrna ◽  
Loop 2

ABSTRACT The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) from purified snRNP proteins and HeLa orXenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts. Substitution of conserved nucleotides comprising internal loop 2 or deletion of internal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing. However, deletion of internal loop 2 abolished U5 activity in splicing and spliceosome formation. Surprisingly, substitution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effect on U5 activity. Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5′ and 3′ halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing. Thus, in contrast to yeast, loop 1 of the human U5 snRNA is dispensable for both steps of splicing in HeLa nuclear extracts. This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins associated with the U5 snRNP. Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution.

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.

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