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PMID: 10024169 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5' splice site.

RNA (New York, N.Y.) ·Vol. 5 ·No. 2 ·1999-02-00 ·Pages 167-79

Reyes JL, Gustafson EH, Luo HR, Moore MJ, Konarska MM

Abstract

A U5 snRNP protein, hPrp8, forms a UV-induced crosslink with the 5' splice site (5'SS) RNA within splicing complex B assembled in trans- as well as in cis-splicing reactions. Both yeast and human Prp8 interact with the 5'SS, branch site, polypyrimidine tract, and 3'SS during splicing. To begin to define functional domains in Prp8 we have mapped the site of the 5'SS crosslink within the hPrp8 protein. Immunoprecipitation analysis limited the site of crosslink to the C-terminal 5060-kDa segment of hPrp8. In addition, size comparison of the crosslink-containing peptides generated with different proteolytic reagents with the pattern of fragments predicted from the hPrp8 sequence allowed for mapping of the crosslink to a stretch of five amino acids in the C-terminal portion of hPrp8 (positions 1894-1898). The site of the 5'SS:hPrp8 crosslink falls within a segment spanning the previously defined polypyrimidine tract recognition domain in yPrp8, suggesting that an overlapping region of Prp8 may be involved both in the 5'SS and polypyrimidine tract recognition events. In the context of other known interactions of Prp8, these results suggest that this protein may participate in formation of the catalytic center of the spliceosome.

MeSH Terms
Amino Acid Sequence Carrier Proteins/genetics,metabolism Cross-Linking Reagents/metabolism Humans Kinetics Molecular Sequence Data Oligoribonucleotides/genetics Peptide Fragments/chemistry RNA Precursors/genetics,metabolism RNA Splicing/genetics RNA-Binding Proteins Ribonucleoprotein, U5 Small Nuclear/genetics,metabolism Spliceosomes/genetics Trypsin/metabolism Ultraviolet Rays
Chemicals
Carrier Proteins Cross-Linking Reagents Oligoribonucleotides PRPF8 protein, human Peptide Fragments RNA Precursors RNA-Binding Proteins Ribonucleoprotein, U5 Small Nuclear Trypsin
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Reyes J L
The Rockefeller University, New York, New York 10021, USA.
Gustafson E H
Luo H R
Moore M J
Konarska M M
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1355-8382
DOI
10.1017/s1355838299981785
Published
1999-02-00
Pages
167-79
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC1369749
Subset
IM
Grants
NIGMS NIH HHS · GM49044 · United States
External Links
PubMed source DOI: 10.1017/s1355838299981785 PMC Full Text View journal details
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