Affinity Purification of Spliceosomal and Small Nuclear Ribonucleoprotein Complexes

2014 ◽  
pp. 957-974
Author(s):  
Julia Dannenberg ◽  
Patrizia Fabrizio ◽  
Cindy L. Will ◽  
Reinhard Lührmann
Keyword(s):  
Affinity Purification ◽  
Ribonucleoprotein Complexes ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

scholarly journals A Novel Yeast U2 snRNP Protein, Snu17p, Is Required for the First Catalytic Step of Splicing and for Progression of Spliceosome Assembly

2001 ◽  
Vol 21 (9) ◽  
pp. 3037-3046 ◽  
Author(s):  
Alexander Gottschalk ◽  
Cornelia Bartels ◽  
Gitte Neubauer ◽  
Reinhard Lührmann ◽  
Patrizia Fabrizio
Keyword(s):  
Affinity Purification ◽  
Rna Recognition Motif ◽  
Spliceosome Assembly ◽  
Exon 2 ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp ◽  
Catalytically Active ◽  
Snrnp Protein ◽  
Nuclear Ribonucleoprotein

ABSTRACT We have isolated and microsequenced Snu17p, a novel yeast protein with a predicted molecular mass of 17 kDa that contains an RNA recognition motif. We demonstrate that Snu17p binds specifically to the U2 small nuclear ribonucleoprotein (snRNP) and that it is part of the spliceosome, since the pre-mRNA and the lariat-exon 2 are specifically coprecipitated with Snu17p. Although the SNU17gene is not essential, its knockout leads to a slow-growth phenotype and to a pre-mRNA splicing defect in vivo. In addition, the first step of splicing is dramatically decreased in extracts prepared from thesnu17 deletion (snu17Δ) mutant. This defect is efficiently reversed by the addition of recombinant Snu17p. To investigate the step of spliceosome assembly at which Snu17p acts, we have used nondenaturing gel electrophoresis. In Snu17p-deficient extracts, the spliceosome runs as a single slowly migrating complex. In wild-type extracts, usually at least two distinct complexes are observed: the prespliceosome, or B complex, containing the U2 but not the U1 snRNP, and the catalytically active spliceosome, or A complex, containing the U2, U6, and U5 snRNPs. Northern blot analysis and affinity purification of the snu17Δ spliceosome showed that it contains the U1, U2, U6, U5, and U4 snRNPs. The unexpected stabilization of the U1 snRNP and the lack of dissociation of the U4 snRNP suggest that loss of Snu17p inhibits the progression of spliceosome assembly prior to U1 snRNP release and after [U4/U6.U5] tri-snRNP addition.

scholarly journals Special Sm Core Complex Functions in Assembly of the U2 Small Nuclear Ribonucleoprotein of Trypanosoma brucei

2009 ◽  
Vol 8 (8) ◽  
pp. 1228-1234 ◽  
Author(s):  
Christian Preußer ◽  
Zsofia Palfi ◽  
Albrecht Bindereif
Keyword(s):  
Trypanosoma Brucei ◽  
Specific Binding ◽  
Core Complex ◽  
Sm Proteins ◽  
Ribonucleoprotein Complexes ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Complex Functions ◽  
Snrnp Protein ◽  
Nuclear Ribonucleoprotein

ABSTRACT The processing of polycistronic pre-mRNAs in trypanosomes requires the spliceosomal small ribonucleoprotein complexes (snRNPs) U1, U2, U4/U6, U5, and SL, each of which contains a core of seven Sm proteins. Recently we reported the first evidence for a core variation in spliceosomal snRNPs; specifically, in the trypanosome U2 snRNP, two of the canonical Sm proteins, SmB and SmD3, are replaced by two U2-specific Sm proteins, Sm15K and Sm16.5K. Here we identify the U2-specific, nuclear-localized U2B″ protein from Trypanosoma brucei. U2B″ interacts with a second U2 snRNP protein, U2-40K (U2A′), which in turn contacts the U2-specific Sm16.5K/15K subcomplex. Together they form a high-affinity, U2-specific binding complex. This trypanosome-specific assembly differs from the mammalian system and provides a functional role for the Sm core variation found in the trypanosomal U2 snRNP.

scholarly journals Characterization of the thermotolerant cell. II. Effects on the intracellular distribution of heat-shock protein 70, intermediate filaments, and small nuclear ribonucleoprotein complexes.

1988 ◽  
Vol 106 (4) ◽  
pp. 1117-1130 ◽  
Author(s):  
W J Welch ◽  
L A Mizzen
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Stress Proteins ◽  
Intracellular Distribution ◽  
Shock Treatment ◽  
Heat Shock Treatment ◽  
Ribonucleoprotein Complexes ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
In Cells

Here we further characterize a number of properties inherent to the thermotolerant cell. In the preceding paper, we showed that the acquisition of the thermotolerant state (by a prior induction of the heat-shock proteins) renders cells translationally tolerant to a subsequent severe heat-shock treatment and thereby results in faster kinetics of both the synthesis and subsequent repression of the stress proteins. Because of the apparent integral role of the 70-kD stress proteins in the acquisition of tolerance, we compared the intracellular distribution of these proteins in both tolerant and nontolerant cells before and after a severe 45 degrees C/30-min shock. In both HeLa and rat embryo fibroblasts, the synthesis and migration of the major stress-induced 72-kD protein into the nucleolus and its subsequent exit was markedly faster in the tolerant cells as compared with the nontolerant cells. Migration of preexisting 72-kD into the nucleolus was shown to be dependent upon heat-shock treatment and independent of active heat-shock protein synthesis. Using both microinjection and immunological techniques, we observed that the constitutive and abundant 73-kD stress protein similarly showed a redistribution from the cytoplasm and nucleus into the nucleolus as a function of heat-shock treatment. We show also that other lesions that occur in cells after heat shock can be prevented or at least minimized if the cells are first made tolerant. Specifically, the heat-induced collapse of the intermediate filament cytoskeleton did not occur in cells rendered thermotolerant. Similarly, the disruption of intranuclear staining patterns of the small nuclear ribonucleoprotein complexes after heat-shock treatment was less apparent in tolerant cells exposed to a subsequent heat-shock treatment.

Human RBM28 protein is a specific nucleolar component of the spliceosomal snRNPs

2006 ◽  
Vol 387 (10/11) ◽  
pp. 1455-1460 ◽  
Author(s):  
Andrey Damianov ◽  
Michael Kann ◽  
William S. Lane ◽  
Albrecht Bindereif
Keyword(s):  
Hela Cell ◽  
Cajal Bodies ◽  
Affinity Selection ◽  
Ribonucleoprotein Complexes ◽  
Small Nuclear Ribonucleoprotein ◽  
Small Nuclear Rnas ◽  
Nucleolar Component ◽  
Nuclear Domains ◽  
Nuclear Ribonucleoprotein ◽  
Selection Of

Abstract The biogenesis of spliceosomal small nuclear RNAs (snRNAs) involves organized translocations between the cytoplasm and certain nuclear domains, such as Cajal bodies and nucleoli. Here we identify human RBM28 protein as a novel snRNP component, based on affinity selection of U6 small nuclear ribonucleoprotein (snRNP). As shown by immunofluorescence, RBM28 is a nucleolar protein. Anti-RBM28 immunoprecipitation from HeLa cell lysates revealed that this protein specifically associates with U1, U2, U4, U5, and U6 snRNAs. Our data provide the first evidence that RBM28 is a common nucleolar component of the spliceosomal ribonucleoprotein complexes, possibly coordinating their transition through the nucleolus.

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