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Vol. 13, No. 5, pp. 581-592, March 1, 1999
Department of Biology, Howard Hughes Medical Institute, Brandeis
University, Waltham, Massachusetts 02454 USA
In the yeast commitment complex and the mammalian E complex, there
is an important base-pairing interaction between the 5' end of U1
snRNA and the conserved 5' splice site region of pre-mRNA. But no
protein contacts between splicing proteins and the pre-mRNA substrate
have been defined in or near this region of early splicing complexes.
To address this issue, we used 4-thiouridine-substituted 5' splice
site-containing RNAs as substrates and identified eight cross-linked
proteins, all of which were identified previously as commitment complex
components. The proteins were localized to three domains: the exon, the
six nucleotides of the 5' ss region, and the downstream intron. The
results indicate that the 5' splice site region and environs are
dense with protein contacts in the commitment complex and suggest that
some of them make important contributions to formation or stability of
the U1 snRNP-pre-mRNA complex.
[Key Words: Yeast commitment complex; 5' splice site; pre-mRNA; 4-thiouridine; cross-link]
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