scholarly journals cDNA cloning of the human U1 snRNA-associated A protein: extensive homology between U1 and U2 snRNP-specific proteins.

1987 ◽  
Vol 6 (12) ◽  
pp. 3841-3848 ◽  
Author(s):  
P. T. Sillekens ◽  
W. J. Habets ◽  
R. P. Beijer ◽  
W. J. van Venrooij
Keyword(s):  
Cdna Cloning ◽  
U2 Snrnp ◽  
U1 Snrna ◽  
Specific Proteins

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 Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions.

1993 ◽  
Vol 13 (1) ◽  
pp. 307-319 ◽  
Author(s):  
S E Behrens ◽  
K Tyc ◽  
B Kastner ◽  
J Reichelt ◽  
R Lührmann
Keyword(s):  
Electron Microscope ◽  
Salt Concentration ◽  
Rnase H ◽  
Globular Domain ◽  
Branch Site ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Specific Proteins ◽  
Molecular Masses ◽  
Nuclear Ribonucleoprotein

Small nuclear (sn) ribonucleoprotein (RNP) U2 functions in the splicing of mRNA by recognizing the branch site of the unspliced pre-mRNA. When HeLa nuclear splicing extracts are centrifuged on glycerol gradients, U2 snRNPs sediment at either 12S (under high salt concentration conditions) or 17S (under low salt concentration conditions). We isolated the 17S U2 snRNPs from splicing extracts under nondenaturing conditions by using centrifugation and immunoaffinity chromatography and examined their structure by electron microscope. In addition to common proteins B', B, D1, D2, D3, E, F, and G and U2-specific proteins A' and B", which are present in the 12S U2 snRNP, at least nine previously unidentified proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa bound to the 17S U2 snRNP. The latter proteins dissociate from the U2 snRNP at salt concentrations above 200 mM, yielding the 12S U2 snRNP particle. Under the electron microscope, the 17S U2 snRNPs exhibited a bipartite appearance, with two main globular domains connected by a short filamentous structure that is sensitive to RNase. These findings suggest that the additional globular domain, which is absent from 12S U2 snRNPs, contains some of the 17S U2-specific proteins. The 5' end of the RNA in the U2 snRNP is more exposed for reaction with RNase H and with chemical probes when the U2 snRNP is in the 17S form than when it is in the 12S form. Removal of the 5' end of this RNA reduces the snRNP's Svedberg value from 17S to 12S. Along with the peculiar morphology of the 17S snRNP, these data indicate that most of the 17S U2-specific proteins are bound to the 5' half of the U2 snRNA.

scholarly journals Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly

2014 ◽  
Vol 28 (22) ◽  
pp. 2518-2531 ◽  
Author(s):  
Shalini Sharma ◽  
Somsakul Pop Wongpalee ◽  
Ajay Vashisht ◽  
James A. Wohlschlegel ◽  
Douglas L. Black
Keyword(s):  
Spliceosome Assembly ◽  
Stem Loop ◽  
U2 Snrnp ◽  
U1 Snrna

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 A 69-kD protein that associates reversibly with the Sm core domain of several spliceosomal snRNP species

1994 ◽  
Vol 124 (3) ◽  
pp. 261-272 ◽  
Author(s):  
W Hackl ◽  
U Fischer ◽  
R Lührmann
Keyword(s):  
Nuclear Transport ◽  
Xenopus Laevis Oocytes ◽  
Sm Proteins ◽  
Core Domain ◽  
Nuclear Extracts ◽  
Core Proteins ◽  
U1 Snrna ◽  
Specific Proteins

The biogenesis of the spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4, and U5 involves: (a) migration of the snRNA molecules from the nucleus to the cytoplasm; (b) assembly of a group of common proteins (Sm proteins) and their binding to a region on the snRNAs called the Sm-binding site; and (c) translocation of the RNP back to the nucleus. A first prerequisite for understanding the assembly pathway and nuclear transport of the snRNPs in more detail is the knowledge of all the snRNP proteins that play essential roles in these processes. We have recently observed a previously undetected 69-kD protein in 12S U1 snRNPs isolated from HeLa nuclear extracts under non-denaturing conditions that is clearly distinct from the U1-70K protein. The following evidence indicates that the 69-kD protein is a common, rather than a U1-specific, protein, possibly associating with the snRNP core particles by protein-protein interaction. (a) Antibodies raised against the 69-kD protein, which did not cross-react with any of the Sm proteins B'-G, precipitated not only U1 snRNPs, but also the other spliceosomal snRNPs U2, U4/U6 and U5, albeit to a lower extent. (b) U1, U2, and U5 core RNP particles reconstituted in vitro contain the 69-kD protein. (c) Xenopus laevis oocytes contain an immunologically related homologue of the human 69-kD protein. When U1 snRNA as well as a mutant U1 snRNA, that can bind the Sm core proteins but lacks the capacity to bind the U1-specific proteins 70K, A, and C, were injected into Xenopus oocytes to allow assembly in vivo, they were recognized by antibodies specific against the 69-kD protein in the ooplasm and in the nucleus. The 69-kD protein is under-represented, if present at all, in purified 17S U2 and in 25S [U4/U6.U5] tri-snRNPs, isolated from HeLa nuclear extracts. Our results are consistent with the working hypothesis that this protein may either play a role in the cytoplasmic assembly of the core domain of the snRNPs and/or in the nuclear transport of the snRNPs. After transport of the snRNPs into the nucleus, it may dissociate from the particles as for example in the case of the 17S U2 or the 25S [U4/U6.U5] tri-snRNP, which bind more than 10 different snRNP specific proteins each in the nucleus.

Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions

1993 ◽  
Vol 13 (1) ◽  
pp. 307-319
Author(s):  
S E Behrens ◽  
K Tyc ◽  
B Kastner ◽  
J Reichelt ◽  
R Lührmann
Keyword(s):  
Electron Microscope ◽  
Salt Concentration ◽  
Rnase H ◽  
Globular Domain ◽  
Branch Site ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Specific Proteins ◽  
Molecular Masses ◽  
Nuclear Ribonucleoprotein

Small nuclear (sn) ribonucleoprotein (RNP) U2 functions in the splicing of mRNA by recognizing the branch site of the unspliced pre-mRNA. When HeLa nuclear splicing extracts are centrifuged on glycerol gradients, U2 snRNPs sediment at either 12S (under high salt concentration conditions) or 17S (under low salt concentration conditions). We isolated the 17S U2 snRNPs from splicing extracts under nondenaturing conditions by using centrifugation and immunoaffinity chromatography and examined their structure by electron microscope. In addition to common proteins B', B, D1, D2, D3, E, F, and G and U2-specific proteins A' and B", which are present in the 12S U2 snRNP, at least nine previously unidentified proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa bound to the 17S U2 snRNP. The latter proteins dissociate from the U2 snRNP at salt concentrations above 200 mM, yielding the 12S U2 snRNP particle. Under the electron microscope, the 17S U2 snRNPs exhibited a bipartite appearance, with two main globular domains connected by a short filamentous structure that is sensitive to RNase. These findings suggest that the additional globular domain, which is absent from 12S U2 snRNPs, contains some of the 17S U2-specific proteins. The 5' end of the RNA in the U2 snRNP is more exposed for reaction with RNase H and with chemical probes when the U2 snRNP is in the 17S form than when it is in the 12S form. Removal of the 5' end of this RNA reduces the snRNP's Svedberg value from 17S to 12S. Along with the peculiar morphology of the 17S snRNP, these data indicate that most of the 17S U2-specific proteins are bound to the 5' half of the U2 snRNA.

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