Document Detail


A general approach for identification of RNA-protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs). Analysis of RNA-protein contacts in native U1 and U4/U6.U5 snRNPs.
MedLine Citation:
PMID:  11006293     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We describe a novel approach to identify RNA-protein cross-linking sites within native small nuclear ribonucleoprotein (snRNP) particles from HeLa cells. It combines immunoprecipitation of the UV-irradiated particles under semi-denaturing conditions with primer extension analysis of the cross-linked RNA moiety. In a feasibility study, we initially identified the exact cross-linking sites of the U1 70-kDa (70K) protein in stem-loop I of U1 small nuclear RNA (snRNA) within purified U1 snRNPs and then confirmed the results by a large-scale preparation that allowed N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry of purified cross-linked peptide-oligonucleotide complexes. We identified Tyr(112) and Leu(175) within the RNA-binding domain of the U1 70K protein to be cross-linked to G(28) and U(30) in stem-loop I, respectively. We further applied our immunoprecipitation approach to HeLa U5 snRNP, as part of purified 25 S U4/U6.U5 tri-snRNPs. Cross-linking sites between the U5-specific 220-kDa protein (human homologue of Prp8p) and the U5 snRNA were located at multiple nucleotides within the highly conserved loop 1 and at one site in internal loop 1 of U5 snRNA. The cross-linking of four adjacent nucleotides indicates an extended interaction surface between loop 1 and the 220-kDa protein. In summary, our approach provides a rapid method for identification of RNA-protein contact sites within native snRNP particles as well as other ribonucleoprotein particles.
Authors:
H Urlaub; K Hartmuth; S Kostka; G Grelle; R Lührmann
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  275     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2000 Dec 
Date Detail:
Created Date:  2001-01-25     Completed Date:  2001-01-26     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  41458-68     Citation Subset:  IM    
Affiliation:
Abteilung Zelluläre Biochemie, Max-Planck-Institut für Biopysikalische Chemie, Am Fassberg 11, D-37077 Göttingen, Germany.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Base Sequence
Humans
Molecular Sequence Data
Precipitin Tests
RNA / chemistry*
Ribonucleoprotein, U1 Small Nuclear / chemistry
Ribonucleoprotein, U4-U6 Small Nuclear / chemistry
Ribonucleoprotein, U5 Small Nuclear / chemistry
Ribonucleoproteins, Small Nuclear / chemistry*
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Spliceosomes / chemistry*
Chemical
Reg. No./Substance:
0/Ribonucleoprotein, U1 Small Nuclear; 0/Ribonucleoprotein, U4-U6 Small Nuclear; 0/Ribonucleoprotein, U5 Small Nuclear; 0/Ribonucleoproteins, Small Nuclear; 63231-63-0/RNA

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