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PMID: 1454055 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

In vitro reconstitution of U1 and U2 snRNPs from isolated proteins and snRNA.

Molecular biology reports ·Vol. 16 ·No. 4 ·1992-09-00 ·Pages 229-40

Sumpter V, Kahrs A, Fischer U, Kornstädt U, Lührmann R

Abstract

In this paper we describe a method for preparing native, RNA-free, proteins from anti-m3G purified snRNPs (U1, U2, U4/U6 and U5) and the subsequent quantitative reconstitution of U1 and U2 snRNPs from purified proteins and snRNA. Reconstituted U1 and U2 snRNPs contained the full complement of core proteins, B, B', D1, D2, D3, E, F and G. Both the U1 and U2 reconstituted particles were stable in CsCl gradients and had the expected buoyant density of 1.4 g/cm3. Reconstituted RNP particle formation was not competited by a 50 fold molar excess of tRNA, as determined by gel retardation assays. However, U1 and U2 particle formation was reduced in the presence of an excess of cold U1 or U2 snRNA demonstrating a specific RNA-protein interaction. U1 and U2 snRNPs were also efficiently reconstituted in vitro, utilizing proteins prepared from mono Q purified U1 and U2 snRNPs. This suggests that for the assembly of snRNPs in vitro no auxiliary proteins other than bona fide snRNP proteins appear to be required. The potential of this reconstitution technique for investigating snRNP assembly and snRNA-protein interactions is discussed.

MeSH Terms
Base Sequence Centrifugation, Density Gradient Chromatography, High Pressure Liquid Molecular Sequence Data RNA, Small Nuclear/chemistry,metabolism Radioimmunoprecipitation Assay Ribonucleoproteins, Small Nuclear/chemistry,isolation & purification,metabolism
Chemicals
RNA, Small Nuclear Ribonucleoproteins, Small Nuclear
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Sumpter V
Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Germany.
Kahrs A
Fischer U
Kornstädt U
Lührmann R
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Article Info
Journal
Molecular biology reports
Abbr.
Mol Biol Rep
ISSN
0301-4851
DOI
10.1007/BF00419662
Published
1992-09-00
Pages
229-40
Language
English
Region
Netherlands
NLM ID
0403234
Subset
IM
External Links
PubMed source DOI: 10.1007/BF00419662 View journal details
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