Academic journal article Genetics

Analysis of Synthetic Lethality Reveals Genetic Interactions between the GTPase Snu114p and snRNAs in the Catalytic Core of the Saccharomyces Cerevisiae Spliceosome

Academic journal article Genetics

Analysis of Synthetic Lethality Reveals Genetic Interactions between the GTPase Snu114p and snRNAs in the Catalytic Core of the Saccharomyces Cerevisiae Spliceosome

Article excerpt

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.

INTRONS in pre-messenger RNA (pre-mRNA) are removed by the spliceosome to yield mature mRNA, which is then exported into the cytoplasm for translation. The process of pre-mRNA splicing has been reviewed extensively (Moore et al. 1993; Will and Lührmann 2006). Splicing is carried out in the nucleus by the assembly of small nuclear ribonucleoprotein particles (or snRNPs) onto the pre-mRNA and occurs by two transesterification steps, which are carried out within the spliceosome by a series of RNA:RNA interactions and RNA:protein rearrangements. Five snRNPs take part in splicing, U1, U2, U4, U5, and U6, of which the last three form a tri-snRNP in which U4 is base paired with U6 (Moore et al. 1993). Although it is thought that the RNAs within snRNPs are responsible for catalyzing the two steps of splicing, it is the many proteins, in particular the NTPases, that bring about the multiple rearrangements that are required for catalysis by the snRNAs (Smith et al. 2008). Key remodelling steps during splicing include unwinding of U4/U6 helices, formation of U2/U6 interactions, unwinding of U2/U6 helices and reestablishment of U4/U6 complexes. Remodelling is facilitated by eight ATPases and one GTPase, Snu114p, one of the key protein factors required for the regulation of spliceosomal dynamics (reviewed in Frazer et al. 2008).

The single GTPase of the spliceosome, Snu114p (U5- 116kDa 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. hSnu114 has been found to crosslink to GTP specifically and a mutation in Snu114p, expected to abolish GTP binding, is lethal, suggesting that GTP binding and hydrolysis are necessary for Snu114p function in vivo (Fabrizio et al. 1997; Bartels et al. 2003). Snu114p also has a unique acidic amino terminus (N terminus) that is implicated, along with the G domain, in U4/U6 unwinding during spliceosome assembly (Bartels et al. 2002).

The relationship of Snu114p to other spliceosomal proteins has been investigated using conditionally lethal alleles to test for genetic interactions with other splicing factors (Brenner and Guthrie 2005). Genetic interactions of snu114 alleles were found with prp8, brr2, prp28, prp19, sad1, and snu66 alleles. The genetic interactions of SNU114 with PRP8 and BRR2 were expected considering the proteins form a stable complex within the U5 snRNPin human cells (Achsel et al. …

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