A dynamic bulge in the U6 RNA internal stem–loop functions in spliceosome assembly and activation

C Joel Mcmanus,Samuel E Butcher,David A Brow,Matthew L Schwartz

doi:10.1261/rna.699907

Abstract

The highly conserved internal stem-loop (ISL) of U6 spliceosomal RNA is unwound for U4/U6 complex formation during spliceosome assembly and reformed upon U4 release during spliceosome activation. The U6 ISL is structurally similar to Domain 5 of group II self-splicing introns, and contains a dynamic bulge that coordinates a Mg++ ion essential for the first catalytic step of splicing. We have analyzed the causes of growth defects resulting from mutations in the Saccharomyces cerevisiae U6 ISL-bulged nucleotide U80 and the adjacent C67-A79 base pair. Intragenic suppressors and enhancers of the cold-sensitive A79G mutation, which replaces the C-A pair with a C-G pair, suggest that it stabilizes the ISL, inhibits U4/U6 assembly, and may also disrupt spliceosome activation. The lethality of mutations C67A and C67G results from disruption of base-pairing potential between U4 and U6, as these mutations are fully suppressed by compensatory mutations in U4 RNA. Strikingly, suppressor analysis shows that the lethality of the U80G mutation is due not only to formation of a stable base pair with C67, as previously proposed, but also another defect. A U6-U80G strain in which mispairing with position 67 is prevented grows poorly and assembles aberrant spliceosomes that retain U1 snRNP and fail to fully unwind the U4/U6 complex at elevated temperatures. Our data suggest that the U6 ISL bulge is important for coupling U1 snRNP release with U4/U6 unwinding during spliceosome activation.

Highlights

Intron removal from nuclear precursor messenger RNAs occurs through a two-step transesterification reaction catalyzed by the spliceosome
To test if the cold-sensitive A79G mutation acts in a fashion similar to A62G, as previously suggested (Fortner et al 1994), we subjected the A79G allele to genetic analysis using two approaches: (1) we randomly mutagenized the U6-A79G allele by error-prone PCR and selected cold-resistant alleles, which are expected to include both revertants and cis-acting suppressors, and (2) we tested the effect of selected suppressors of A62G in cis with A79G
A79C and A79U were synthetic lethal with C67U, even though the resulting base pairs should not be stable. These results indicate that internal stem–loop (ISL) bulge residues function cooperatively, and the growth defects of mutations in positions 67 and 79 are not due to the formation of a stable base pair

Summary

Introduction

Intron removal from nuclear precursor messenger RNAs (pre-mRNAs) occurs through a two-step transesterification reaction catalyzed by the spliceosome. The spliceosome is a multimegadalton complex consisting primarily of five small nuclear ribonucleoprotein particles, or snRNPs, named U1, U2, U4, U5, and U6 for their respective small nuclear RNA (snRNA) component. These snRNPs bind to an intron sequentially: first U1, U2, the U4/U5/U6 trisnRNP complex. The spliceosome is activated through a number of structural rearrangements in the snRNAs, facilitated by protein components (Will and Luhrmann 2006) These rearrangements result in the coordinated dissociation of U1 and U4 snRNPs (Kuhn et al 1999; Staley and Guthrie 1999), during which U6 becomes.

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