A 3'-Terminal Minihelix in the Precursor of Human Spliceosomal U2
Small Nuclear RNA*
Annie
Mougin
§,
Françoise
Torterotot,
Christiane
Branlant,
Marty R.
Jacobson¶
,
Qian
Huang¶**, and
Thoru
Pederson¶
From the Unité Mixte Recherche 7567 CNRS-Université Henri Poincaré Nancy I, Maturation des ARN
et Enzymologie Moléculaire, Université H. Poincaré,
54506 Vandoeuvre-les Nancy, France and the ¶ Department of
Biochemistry and Molecular Pharmacology, University of Massachusetts
Medical School, Worcester, Massachusetts 01605
U2 RNA is one of five small nuclear
RNAs that participate in the majority of mRNA splicing. In addition
to its role in mRNA splicing, the biosynthesis of U2 RNA and three
of the other spliceosomal RNAs is itself an intriguing process
involving nuclear export followed by 5'-cap hypermethylation, assembly
with specific proteins, 3' end processing, and then nuclear import.
Previous work has identified sequences near the 3' end of pre-U2 RNA
that are required for accurate and efficient processing. In this study,
we have investigated the structural basis of U2 RNA 3' end processing by chemical and enzymatic probing methods. Our results demonstrate that
the 3' end of pre-U2 RNA is a minihelix with an estimated stabilization
free energy of 
6.9 kcal/mol. Parallel RNA structure mapping
experiments with mutant pre-U2 RNAs revealed that the presence of this
3' minihelix is itself not required for in vitro 3'-processing of pre-U2 RNA, in support of earlier studies implicating internal regions of pre-U2 RNA. Other considerations raise the possibility that this distinctive structural motif at the 3' end of
pre-U2 RNA plays a role in the cleavage of the precursor from its
longer primary transcript or in its nucleocytoplasmic traffic.
*
This work was supported by grants from the French Centre
National de la Recherche Scientifique and the French Ministère de la Recherche et de l'Enseignement Supérieure (to A. M. and
C. B.) and National Institutes of Health Grant GM-21595 (to T. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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