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 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 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.

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 Demonstration of a dynamic, transcription-dependent organization of pre-mRNA splicing factors in polytene nuclei.

1996 ◽  
Vol 133 (5) ◽  
pp. 929-941 ◽  
Author(s):  
G Baurén ◽  
W Q Jiang ◽  
K Bernholm ◽  
F Gu ◽  
L Wieslander
Keyword(s):  
Mrna Splicing ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Chironomus Tentans ◽  
Transcription Inhibition ◽  
Physiological Conditions ◽  
Active Gene ◽  
U2 Snrnp ◽  
Tight Linkage ◽  
Polytene Nuclei

We describe the dynamic organization of pre-mRNA splicing factors in the intact polytene nuclei of the dipteran Chironomus tentans. The snRNPs and an SR non-snRNP splicing factor are present in excess, mainly distributed throughout the interchromatin. Approximately 10% of the U2 snRNP and an SR non-snRNP splicing factor are associated with the chromosomes, highly enriched in active gene loci where they are bound to RNA. We demonstrate that the splicing factors are specifically recruited to a defined gene upon induction of transcription during physiological conditions. Concomitantly, the splicing factors leave gene loci in which transcription is turned off. We also demonstrated that upon general transcription inhibition, the splicing factors redistribute from active gene loci to the interchromatin. Our findings demonstrate the dynamic intranuclear organization of splicing factors and a tight linkage between transcription and the intranuclear organization of the splicing machinery.

Differential block of U small nuclear ribonucleoprotein particle interactions during in vitro splicing of adenovirus E1A transcripts containing abnormally short introns

1991 ◽  
Vol 11 (3) ◽  
pp. 1258-1269
Author(s):  
M Himmelspach ◽  
R Gattoni ◽  
C Gerst ◽  
K Chebli ◽  
J Stévenin
Keyword(s):  
Donor Site ◽  
Ribonucleoprotein Particle ◽  
Adenovirus E1a ◽  
Branch Site ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
The Common ◽  
Nuclear Ribonucleoprotein

We have studied the consequences of decreasing the donor site-branch site distance on splicing factor-splice site interactions by analyzing alternative splicing of adenovirus E1A pre-mRNAs in vitro. We show that the proximal 13S donor site has a cis-inhibiting effect on the 9S and 12S mRNA reactions when it is brought too close to the common branch site, suggesting that the factor interactions in the common 3' part of the intron are impaired by the U1 small nuclear ribonucleoprotein particle (snRNP) binding to the displaced 13S donor site. Further analysis of the interactions was carried out by studying complex assembly and the accessibility to micrococcal nuclease digestion of 5'-truncated E1A substrates containing only splice sites for the 13S mRNA reaction. A deletion which brings the donor site- branch site distance to 49 nucleotides, which is just below the minimal functional distance, results in a complete block of the U4-U5-U6 snRNP binding, whereas a deletion 15 nucleotides larger results in a severe inhibition of the formation of the U2 snRNP-containing complexes. Sequence accessibility analyses performed by using the last mini-intron-containing transcript demonstrate that the interactions of U2 snRNP with the branch site are strongly impaired whereas the initial bindings of U1 snRNP to the donor site and of specific factors to the 3' splice site are not significantly modified. Our results strongly suggest that the interaction of U1 snRNP with the donor site of a mini-intron is stable enough in vitro to affect the succession of events leading to U2 snRNP binding with the branch site.

scholarly journals Nuclear exchange of the U1 and U2 snRNP-specific proteins.

1990 ◽  
Vol 110 (4) ◽  
pp. 871-881 ◽  
Author(s):  
R J Feeney ◽  
G W Zieve
Keyword(s):  
Common Core ◽  
Kinetic Studies ◽  
Isotopic Labeling ◽  
Cell Fractionation ◽  
Isolated Nuclei ◽  
Core Proteins ◽  
U2 Snrnp ◽  
Specific Proteins ◽  
The Common ◽  
Nuclear Exchange

The snRNP particles include a set of common core snRNP proteins and snRNP specific proteins. In rodent cells the common core proteins are the B, D, D', E, F and G proteins in a suggested stoichiometry of B2D'2D2EFG. The additional U1- and U2-specific proteins are the 70-kD, A and C proteins and the A' and B" proteins, respectively. Previous cell fractionation and kinetic analysis demonstrated the snRNP core proteins are stored in the cytoplasm in large partially assembled snRNA-free intermediates that assemble with newly synthesized snRNAs during their transient appearance in the cytoplasm (Sauterer, R. A., R. J. Feeney, and G. W. Zieve. 1988. Exp. Cell Res. 176:344-359). This report investigates the assembly and intracellular distribution of the U1 and U2 snRNP-specific proteins. Cell enucleation and aqueous cell fractionation are used to prepare nuclear and cytoplasmic fractions and the U1- and U2-specific proteins are identified by isotopic labeling and immunoprecipitation or by immunoblotting with specific autoimmune antisera. The A, C, and A' proteins are found both assembled into mature nuclear snRNP particles and in unassembled pools in the nucleus that exchange with the assembled snRNP particles. The unassembled proteins leak from isolated nuclei prepared by detergent extraction. The unassembled A' protein sediments at 4S-6S in structures that may be multimers. The 70-kD and B" proteins are fully assembled with snRNP particles which do not leak from isolated nuclei. The kinetic studies suggest that the B" protein assembles with the U2 particle in the cytoplasm before it enters the nucleus.

scholarly journals Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Andrea Pawellek ◽  
Ursula Ryder ◽  
Triin Tammsalu ◽  
Lewis J King ◽  
Helmi Kreinin ◽  
...  
Keyword(s):  
Complex Formation ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
E Coli ◽  
Sumo Protease ◽  
Complex Cells ◽  
U2 Snrnp ◽  
In Vitro Splicing ◽  
In Cells

We have identified the plant biflavonoid hinokiflavone as an inhibitor of splicing in vitro and modulator of alternative splicing in cells. Chemical synthesis confirms hinokiflavone is the active molecule. Hinokiflavone inhibits splicing in vitro by blocking spliceosome assembly, preventing formation of the B complex. Cells treated with hinokiflavone show altered subnuclear organization specifically of splicing factors required for A complex formation, which relocalize together with SUMO1 and SUMO2 into enlarged nuclear speckles containing polyadenylated RNA. Hinokiflavone increases protein SUMOylation levels, both in in vitro splicing reactions and in cells. Hinokiflavone also inhibited a purified, E. coli expressed SUMO protease, SENP1, in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased in cells following hinokiflavone treatment, with the major targets including six proteins that are components of the U2 snRNP and required for A complex formation.

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 SRPK2: A Differentially Expressed SR Protein-specific Kinase Involved in Mediating the Interaction and Localization of Pre-mRNA Splicing Factors in Mammalian Cells

1998 ◽  
Vol 140 (4) ◽  
pp. 737-750 ◽  
Author(s):  
Huan-You Wang ◽  
Wen Lin ◽  
Jacqueline A. Dyck ◽  
Joanne M. Yeakley ◽  
Zhou Songyang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Splicing Regulation ◽  
Phosphorylation Sites ◽  
Sr Protein ◽  
Random Peptide ◽  
Selection For

Abstract. Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain–containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain–containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.

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