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. Copyright © 2009 by the Genetics Society of America.