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PMID: 9740126 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Probing of the spliceosome with site-specifically derivatized 5' splice site RNA oligonucleotides.

RNA (New York, N.Y.) ·Vol. 4 ·No. 9 ·1998-09-00 ·Pages 1069-82

Sha M, Levy T, Kois P, Konarska MM

Abstract

We have developed a site-specific chemical modification technique to incorporate a photoreactive azidophenacyl (APA) group at designated internal positions along the RNA phosphodiester backbone. Using this technique, we have analyzed interactions of the 5' splice site (5'SS) RNA within the spliceosome. Several crosslinked products can be detected within complex B using the derivatized 5'SS RNAs, including U6 snRNA, hPrp8p, and 114-, 90-, 70-, 54-, and 27-kDa proteins. The 5'SS RNAs derivatized at intron positions +4 to +8 crosslink to U6 snRNA, confirming the previously reported pairing interaction between these sequences. hPrp8p and p70 are crosslinked to the 5'SS RNA when the APA is placed within the 5' exon. Finally, a set of unidentified proteins, including p114, p54, and p27, is detected with the 5'SS RNA derivatized at intron positions +4 to +8. Introduction of the bulky APA group near the 5'SS junction (positions -2 to +3) strongly interferes with complex B formation and thus no APA crosslinks are observed at these positions. Together with our earlier observation that hPrp8p crosslinks to the GU dinucleotide at the 5' end of the intron, these results suggest that the inhibitory effect of APA results from steric hindrance of the hPrp8p:5'SS interaction. Unexpectedly, thio-modifications within the region of the 5'SS RNA that is involved in base pairing to U6 snRNA strongly stimulate complex B formation.

MeSH Terms
Allosteric Site Animals Azides Base Composition Base Sequence Binding Sites Cross-Linking Reagents Humans In Vitro Techniques Introns Oligoribonucleotides/chemistry,genetics,metabolism Photochemistry RNA Splicing RNA, Small Nuclear/chemistry,genetics,metabolism Spliceosomes/genetics,metabolism
Chemicals
Azides Cross-Linking Reagents Oligoribonucleotides RNA, Small Nuclear
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Sha M
The Rockefeller University, New York, New York 10021, USA.
Levy T
Kois P
Konarska M M
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1355-8382
DOI
10.1017/s1355838298980682
Published
1998-09-00
Pages
1069-82
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC1369683
Subset
IM
External Links
PubMed source DOI: 10.1017/s1355838298980682 PMC Full Text View journal details
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