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PMID: 1740113 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Reconstituted mammalian U4/U6 snRNP complements splicing: a mutational analysis.

The EMBO journal ·Vol. 11 ·No. 1 ·1992-01-00 ·Pages 345-59

Wolff T, Bindereif A

Abstract

We have developed an in vitro complementation assay to analyse the functions of U6 small nuclear RNA (snRNA) in splicing and in the assembly of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. U6-specific, biotinylated 2'-OMe RNA oligonucleotides were used to deplete nuclear extract of the U4/U6 snRNP and to affinity purify functional U4 snRNP. The addition of affinity purified U4 snRNP together with U6 RNA efficiently restored splicing activity, spliceosome assembly and U4/U5/U6 multi-snRNP formation in the U4/U6-depleted extract. Through a mutational analysis we have obtained evidence for multiple sequence elements of U6 RNA functioning during U4/U5/U6 multi-snRNP formation, spliceosome assembly and splicing. Surprisingly, the entire 5' terminal domain of U6 RNA is dispensable for splicing function. In contrast, two regions in the central and 3' terminal domain are required for the assembly of a functional U4/U5/U6 multi-snRNP. Another sequence in the 3' terminal domain plays an essential role in spliceosome assembly; a model is strongly supported whereby base pairing between this sequence and U2 RNA plays an important role during assembly of a functional spliceosome.

MeSH Terms
Bacterial Proteins/metabolism Base Sequence Cell Nucleus/metabolism Chromatography, Affinity DNA Mutational Analysis Genetic Complementation Test HeLa Cells Humans Models, Molecular Molecular Sequence Data Nucleic Acid Conformation RNA Splicing RNA, Small Nuclear/genetics,isolation & purification,metabolism Streptavidin Subcellular Fractions
Chemicals
Bacterial Proteins RNA, Small Nuclear Streptavidin
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Wolff T
Max-Planck-Institut für Molekulare Genetik, Otto-Warburg-Laboratorium, Berlin, Dahlem, FRG.
Bindereif A
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1992-01-00
Pages
345-59
Language
English
Region
England
NLM ID
8208664
PMCID
PMC556455
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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