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PMID: 9303319 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Evidence that U5 snRNP recognizes the 3' splice site for catalytic step II in mammals.

The EMBO journal ·Vol. 16 ·No. 15 ·1997-08-01 ·Pages 4746-59

Chiara MD, Palandjian L, Feld Kramer R, Reed R

Abstract

The first AG dinucleotide downstream from the branchpoint sequence (BPS) is chosen as the 3' splice site during catalytic step II of the splicing reaction. The mechanism and factors involved in selection of this AG are not known. Early in mammalian spliceosome assembly, U2AF65 binds to the pyrimidine tract between the BPS and AG. Here we show that U2AF65 crosslinking is replaced by crosslinking of three proteins of 110, 116 and 220 kDa prior to catalytic step II, and we provide evidence that all three proteins are components of U5 snRNP. These proteins interact with pre-mRNA in the region spanning from immediately downstream of U2 snRNP's binding site at the BPS to just beyond the 3' splice site. We also demonstrate that there are strict constraints on both the sequence and the distance between the BPS and AG for catalytic step II. Together, these observations suggest that U5 snRNP is positioned on the 3' splice site by an interaction (direct or indirect) with U2 snRNP bound at the BPS and by a direct interaction with the pyrimidine tract. The functional AG for catalytic step II may be specified, in turn, by its location with respect to the U5 snRNP binding site.

MeSH Terms
Animals Base Sequence Binding Sites Cross-Linking Reagents Kinetics Mammals Models, Biological Molecular Sequence Data Protein Binding RNA Precursors/genetics,metabolism RNA Splicing RNA-Binding Proteins/isolation & purification,metabolism Ribonucleoprotein, U5 Small Nuclear/metabolism Saccharomyces cerevisiae/metabolism Spliceosomes/metabolism
Chemicals
Cross-Linking Reagents RNA Precursors RNA-Binding Proteins Ribonucleoprotein, U5 Small Nuclear
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Chiara M D
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Palandjian L
Feld Kramer R
Reed R
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43 references, click to expand
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
DOI
10.1093/emboj/16.15.4746
Published
1997-08-01
Pages
4746-59
Language
English
Region
England
NLM ID
8208664
PMCID
PMC1170101
Subset
IM
External Links
PubMed source DOI: 10.1093/emboj/16.15.4746 PMC Full Text View journal details
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