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PMID: 11604503 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Dual function for U2AF(35) in AG-dependent pre-mRNA splicing.

Molecular and cellular biology ·Vol. 21 ·No. 22 ·2001-11-00 ·Pages 7673-81

Guth S, Tange TØ, Kellenberger E, Valcárcel J

Abstract

The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF(65)) binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF(35)) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U2AF(35) and the 3' splice site AG can stabilize U2AF(65) binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF(35) has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF(65) binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF(65). In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF(35) dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF(35) dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF(35) function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF(35) activity and because a truncation mutant of U2AF(35) consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF(35) can be explained in terms of enhanced U2AF(65) binding, other activities of U2AF(35) do not correlate with increased cross-linking of U2AF(65) to the Py tract. Collectively, the results argue that interaction of U2AF(35) with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF(65) binding, thus revealing a dual function for U2AF(35) in pre-mRNA splicing.

MeSH Terms
Binding Sites Chromosome Mapping Dinucleotide Repeats HeLa Cells Humans Immunoglobulin M/genetics Nuclear Proteins RNA Precursors RNA Splicing Ribonucleoproteins/metabolism,physiology Splicing Factor U2AF
Chemicals
Immunoglobulin M Nuclear Proteins RNA Precursors Ribonucleoproteins Splicing Factor U2AF U2AF1 protein, human U2AF2 protein, human Zrsr1 protein, mouse
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Guth S
Gene Expression Programme, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
Tange T Ø
Kellenberger E
Valcárcel J
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.21.22.7673-7681.2001
Published
2001-11-00
Pages
7673-81
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC99938
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.21.22.7673-7681.2001 PMC Full Text View journal details
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