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Science 24 December 1993:
Vol. 262. no. 5142, pp. 1989 - 1996
DOI: 10.1126/science.8266094
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Articles

Science, Vol 262, Issue 5142, 1989-1996
Copyright © 1993 by American Association for the Advancement of Science

articles

The U5 and U6 small nuclear RNAs as active site components of the spliceosome

EJ Sontheimer and JA Steitz

Howard Hughes Medical Institute, Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT 06536-0812.

Five small nuclear RNAs (U1, U2, U4, U5, and U6) participate in precursor messenger RNA (pre-mRNA) splicing. To probe their interactions within the active center of the mammalian spliceosome, substrates containing a single photoactivatable 4-thiouridine residue adjacent to either splice site were synthesized, and crosslinks were induced during the course of in vitro splicing. An invariant loop sequence in U5 small nuclear RNA contacts exon 1 before and after the first step of splicing because a crosslink between U5 and the last residue of exon 1 appeared in the pre-mRNA and then in the cutoff exon 1 intermediate. Both of these crosslinked species could undergo subsequent splicing, indicating that the crosslinks reflect a functional interaction that is maintained through both reaction steps. The same U5 loop aligns the two exons for ligation since the first residue of exon 2 also became crosslinked to U5 in the lariat intermediate. An invariant sequence in U6 RNA became crosslinked to the conserved second position of the intron within both the lariat intermediate and the lariat intron product. On the basis of these results, several conformational arrangements of small nuclear RNAs within the spliceosomal active center can be distinguished, and additional mechanistic parallels between the spliceosome and self-splicing introns can be drawn.


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Guides to the heart of the spliceosome.
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