Academic journal article Genetics

Genetic Analysis Reveals a Role for the C Terminus of the Saccharomyces Cerevisiae GTPase Snu114 during Spliceosome Activation

Academic journal article Genetics

Genetic Analysis Reveals a Role for the C Terminus of the Saccharomyces Cerevisiae GTPase Snu114 during Spliceosome Activation

Article excerpt

ABSTRACT

Snu114 is the only GTPase required for mRNA splicing. As a homolog of elongation factor G, it contains three domains (III-V) predicted to undergo a large rearrangement following GTP hydrolysis. To assess the functional importance of the domains of Snu114, we used random mutagenesis to create conditionally lethal alleles. We identified three main classes: (1) mutations that are predicted to affect GTP binding and hydrolysis, (2) mutations that are clustered in 10- to 20-amino-acid stretches in each of domains III-V, and (3) mutations that result in deletion of up to 70 amino acids from the C terminus. Representative mutations from each of these classes blocked the first step of splicing in vivo and in vitro. The growth defects caused by most alleles were synthetically exacerbated by mutations in PRP8, a U5 snRNP protein that physically interacts with Snu114, as well as in genes involved in snRNP biogenesis, including SAD1 and BRR1. The allele snu114-60, which truncates the C terminus, was synthetically lethal with factors required for activation of the spliceosome, including the DExD/H-box ATPases BRR2 and PRP28. We propose that GTP hydrolysis results in a rearrangement between Prp8 and the C terminus of Snu114 that leads to release of U1 and U4, thus activating the spliceosome for catalysis.

PRE-mRNA splicing is catalyzed by the spliceosome, a large dynamic complex composed of five small nuclear RNAs (snRNAs) and >80 proteins (BuRGE et al 1998; JURICA and MOORE 2003). The chemistry of splicing comprises two sequential transesterification reactions (MOORE et al. 1993). In the first reaction, the 5' splice site is cleaved and a branched lariat structure is formed within the intron. In the second reaction, the 3' splice site is cleaved and the two exons are joined together. During the splicing cycle, the RNA and protein components of the spliceosome undergo numerous rearrangements, which must be highly coordinated to ensure fidelity of the process (STALEY and GUTHRIE 1998). Most of these rearrangements appear to be energy dependent and are correlated with the activity of individual ATPases of the DExD/H-box family. Eight known DExD/H-box proteins are required for the splicing cycle, and mutations in these proteins inhibit the ATP-dependent steps of splicing (STALEY and GUTHRIE 1998). Additionally, splicing requires one GTPase, Snulle, which is an essential protein in Saccharomyces cerevisiae (FABRizio et al. 1997). Notably, Snullé is homologous to the ribosomal translocase elongation factor G (EF-G in prokaryotes/EF2 in eukaryotes), leading to the hypothesis that Snulle may similarly use the energy of GTP hydrolysis to drive rearrangements of the spliceosome (FABRIZIO et al. 1997).

Snullé is packaged with other proteins and the U5 snRNA to form the U5 small ribonucleoprotein particle (snRNP). Prior to formation of the spliceosome, U5 snRNP interacts with the U4/U6 di-snRNP, in which U4 and U6 snRNAs are extensively base paired, thus forming U4/U6-U5 tri-snRNP (reviewed in BURGE et al. 1998). According to the canonical model of spliceosome assembly, the tri-snRNP is then recruited to the prespliceosome, in which Ul snRNA is base paired with the 5' splice site and U2 snRNA is base paired with the branchpoint sequence, an intronic consensus sequence near the 3' splice site. Although the addition of trisnRNP forms the complete spliceosome, this complex is catalytically inert. Activation requires that the Ul/5' splice site interaction and the base pairing between U4 and U6 be disrupted, such that Ul and U4 are no longer stably associated with the spliceosome. In contrast to the stepwise pathway of spliceosome assembly, recent evidence suggests that a holospliceosome containing all five snRNPs interacts as a complex with each intron (STEVENS et al. 2002). Nonetheless, ordered rearrangements of the snRNPs must occur prior to catalysis.

Rearrangements that occur during the early stages of spliceosome activation are regulated by several components of the U5 snRNP (BROW 2002). …

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