Immunoaffinity Purification of Spliceosomal and Small Nuclear Ribonucleoprotein Complexes

2008 ◽  
pp. 694-709 ◽  
Author(s):  
Cindy L. Will ◽  
Evgeny M. Makarov ◽  
Olga V. Makarova ◽  
Reinhard Lhrmann
Keyword(s):  
Ribonucleoprotein Complexes ◽  
Small Nuclear Ribonucleoprotein ◽  
Immunoaffinity Purification ◽  
Nuclear Ribonucleoprotein

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.

scholarly journals Smn, the spinal muscular atrophy–determining gene product, modulates axon growth and localization of β-actin mRNA in growth cones of motoneurons

2003 ◽  
Vol 163 (4) ◽  
pp. 801-812 ◽  
Author(s):  
Wilfried Rossoll ◽  
Sibylle Jablonka ◽  
Catia Andreassi ◽  
Ann-Kathrin Kröning ◽  
Kathrin Karle ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Gene Product ◽  
Muscular Atrophy ◽  
Axon Growth ◽  
Growth Cones ◽  
Binding Partner ◽  
Ribonucleoprotein Complexes ◽  
Small Nuclear Ribonucleoprotein ◽  
Actin Mrna ◽  
Nuclear Ribonucleoprotein

Spinal muscular atrophy (SMA), a common autosomal recessive form of motoneuron disease in infants and young adults, is caused by mutations in the survival motoneuron 1 (SMN1) gene. The corresponding gene product is part of a multiprotein complex involved in the assembly of spliceosomal small nuclear ribonucleoprotein complexes. It is still not understood why reduced levels of the ubiquitously expressed SMN protein specifically cause motoneuron degeneration. Here, we show that motoneurons isolated from an SMA mouse model exhibit normal survival, but reduced axon growth. Overexpression of Smn or its binding partner, heterogeneous nuclear ribonucleoprotein (hnRNP) R, promotes neurite growth in differentiating PC12 cells. Reduced axon growth in Smn-deficient motoneurons correlates with reduced β-actin protein and mRNA staining in distal axons and growth cones. We also show that hnRNP R associates with the 3′ UTR of β-actin mRNA. Together, these data suggest that a complex of Smn with its binding partner hnRNP R interacts with β-actin mRNA and translocates to axons and growth cones of motoneurons.

scholarly journals Precursors of U4 small nuclear RNA.

1984 ◽  
Vol 99 (3) ◽  
pp. 1140-1144 ◽  
Author(s):  
S J Madore ◽  
E D Wieben ◽  
G R Kunkel ◽  
T Pederson
Keyword(s):  
Small Nuclear Rna ◽  
Rna Sequences ◽  
Pulse Label ◽  
Ribonucleoprotein Complexes ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Rna ◽  
Labeled Rna ◽  
U4 Rna ◽  
Nuclear Ribonucleoprotein ◽  
Cytoplasmic Rnas

The processing and ribonucleoprotein assembly of U4 small nuclear RNA has been investigated in HeLa cells. After a 45-min pulse label with [3H]uridine, a set of apparently cytoplasmic RNAs was observed migrating just behind the gel electrophoretic position of mature U4 RNA. These molecules were estimated to be one to at least seven nucleotides longer than mature U4 RNA. They reacted with Sm autoimmune patient sera and a monoclonal Sm antibody, indicating their association with proteins characteristic of small nuclear ribonucleoprotein complexes. The same set of RNAs was identified by hybrid selection of pulse-labeled RNA with cloned U4 DNA, confirming that these are U4 RNA sequences. No larger nuclear precursors of these RNAs were detected. Pulse-chase experiments revealed a progressive decrease in the radioactivity of the U4 precursor RNAs coincident with an accumulation of labeled mature U4 RNA, confirming a precursor-product relationship.

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