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Abstract
Conformational changes of snRNAs in the spliceosome required for pre-mRNA splicing are regulated by eight ATPases and one GTPase Snu114p. The Snu114p guanine state regulates U4/U6 unwinding during spliceosome activation and U2/U6 unwinding during spliceosome disassembly through the ATPase Brr2p. We investigated 618 genetic interactions to identify an extensive genetic interaction network between SNU114 and snRNAs. Snu114p G domain alleles were exacerbated by mutations that stabilize U4/U6 base pairing. G domain alleles were made worse by U2 and U6 mutations that stabilize or destabilize U2/U6 base pairing in helix I. Compensatory mutations that restored U2/U6 base pairing in helix I relieved synthetic lethality. Snu114p G domain alleles were also worsened by mutations in U6 predicted to increase 5' splice site base pairing. Both N-terminal and G domain alleles were exacerbated by U5 loop 1 mutations at positions involved in aligning exons while C-terminus alleles were synthetically lethal with U5 internal loop 1 mutations. This suggests a spatial orientation for Snu114p with U5. We propose that the RNA base pairing state is directly or indirectly sensed by the Snu114p G domain allowing the Snu114p C-terminal domain to regulate Brr2p or other proteins to bring about RNA/RNA rearrangements required for splicing.
Highlights
Conformational changes of snRNAs in the spliceosome required for pre-mRNA splicing are regulated by eight ATPases and one GTPase Snu114p
Snu114 alleles are annotated with their putative positions within Snu114p, for example, V266P (G4) signifies the mutation is in G domain motif G4 and P860K (IVa) signifies the mutation is in domain IVa
Transformants were transferred to 5-fluoroorotic acid (5-FOA) to evict the URA3 plasmid containing the wild-type genes and determine the synthetic lethality between mutant alleles
Summary
Conformational changes of snRNAs in the spliceosome required for pre-mRNA splicing are regulated by eight ATPases and one GTPase Snu114p. Snu114p G domain alleles were worsened by mutations in U6 predicted to increase 59 splice site base pairing Both N-terminal and G domain alleles were exacerbated by U5 loop 1 mutations at positions involved in aligning exons while C-terminus alleles were synthetically lethal with U5 internal loop 1 mutations. We propose that the RNA base pairing state is directly or indirectly sensed by the Snu114p G domain allowing the Snu114p C-terminal domain to regulate Brr2p or other proteins to bring about RNA/RNA rearrangements required for splicing. The single GTPase of the spliceosome, Snu114p (U5116kDa or hSnu114 in humans), is a protein of the U5 snRNP (Fabrizio et al 1997) It shares significant sequence homology with translation elongation factor 2 (EF-2) and contains five domains similar to EF-2, with domain I containing the G domain consensus sequence elements G1–G5 that are important for binding and hydrolysis of GTP. The genetic interactions of SNU114 with PRP8 and BRR2 were expected considering the proteins form a stable complex
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