Discussion
The present study provides new genetic insights to the impact of the lack of TMG caps in budding yeast. Prior screening for synthetic lethal and sick tgs1∆ interactions had drawn attention specifically to the U1 snRNP as a focal point for TMG cap function in vivo (Hausmann et al. 2008). This idea was fortified by the findings that the only overt change in the composition of yeast spliceosomal snRNPs in tgs1∆ cells was the gain of CBC as a stoichiometric component of the U1 snRNP, by virtue of its binding to the residual m7G cap on the U1 snRNA (Schwer et al. 2011). In TGS1 cells, CBC is loosely associated with the U1 snRNP at low sub-stoichiometric levels compared to the intrinsic U1 snRNP subunits (Schwer et al. 2011). It is thought that CBC interacts with one or more of the U1 snRNP proteins to facilitate bridging interactions between CBC bound to the pre-mRNA m7G cap and the U1 snRNP at the 5′ splice site (Lewis et al. 1996; Görnemann et al. 2005). Hypomorphic mutations in Cbc2 that weaken cap binding suppress the tgs1∆ cs growth defect (Qiu et al. 2012).
Here, we show that restoration of growth of tgs1∆ cells in the cold can also be achieved by deleting the C-terminal 77-aa segment of the essential Snp1 subunit of the U1 snRNP. Although it had been appreciated earlier that this C-terminal portion of Snp1 was dispensable for vegetative growth (Hilleren et al. 1995), the genetic interactions of the Snp1-C∆ truncations were not interrogated. Underscoring the theme of redundancy in the yeast U1 snRNP, we show that otherwise benign Snp1-(1-223) and Snp1-(1-208) mutations are catastrophic in the absence of Mud2 or Nam8. Yet these same SNP1 truncation alleles elicit a gain-of-function in the tgs1∆ genetic background. Our frugal speculation is that tgs1∆ suppression by SNP1-C∆ is mediated via an effect on CBC association with the residual U1 snRNA m7G cap, whereby the C-terminal segment of Snp1 is itself a point of contact of CBC with the U1 snRNP. In this scenario, weakening of the CBC•U1 snRNP interaction by Snp1 truncation would diminish CBC association with the U1 m7G cap in tgs1∆ cells and allow growth in the cold (more or less mimicking the Cbc2 cap-binding site mutants with respect to tgs1∆ suppression). In TGS1 cells that have TMG caps, the Cbc2 cap-binding site lesion Y24A and the Snp1-C∆ truncations, which cause no growth defects per se, synergized when combined to mimic the severe cold sensitivity of the cbc2∆ null mutant. These findings are consistent with the idea that the Snp1 C-terminus contributes to the interaction of CBC with the U1 snRNP.
The C-terminal 77-aa segment of yeast Snp1 is rich in arginine (n = 11), serine (n = 12), and alanine (n = 11) and is predicted to be strongly hydrophilic, with the exception of one hydrophobic tract (265PLLSAATPTAAVTSVY280). The amino acid sequence and composition are suggestive of structural disorder or a structure that is templated by the association of this polypeptide with other proteins. Because this segment is not conserved in human U1-70K and the C-terminus of U1-70K is not seen in U1 snRNP crystal structures, we cannot intuit what contacts might be made by the Snp1 C-terminus. This will be an interesting topic for future studies given the broad impact, both positive and negative, of Snp1 C-terminal deletion on yeast physiology when other components of the splicing apparatus are simultaneously perturbed.
In a separate approach entailing a genomic library screen, we identified RPO26 as a dosage suppressor of the cold-sensitive phenotype of tgs1∆ cells. Because even a nominally single extra copy of the RPO26 gene on a CEN plasmid revived tgs1∆ growth at 18°, we surmise that RPO26 is an especially vulnerable target of the effect of the tgs1∆ mutation. This vulnerability is unlikely to reflect a fastidious gene-specific requirement for TMG caps or other splicing factors in removing the RPO26 intron, insofar as the RPO26 5′-splice site, branchpoint, and 3′-splice site adhere perfectly to the yeast consensus sequences and the intron is situated close to the 5′ end of the RPO26 ORF, as is the case for most yeast genes. Rather, it is the fact that even small changes in RPO26 expression can result in an overt growth defect in the cold (Nouraini et al. 1996b) that allowed us to recover a singularly sensitive target gene in the suppressor screen.
As discussed above, we implicate ectopic binding of nuclear CBC to the m7G cap of the U1 snRNP of tgs1∆ cells as a principal factor in the cold sensitivity of tgs1∆ cells. Mutations in the cap-binding site of CBC or deletion of the Snp1 C-terminus completely restore normal growth of tgs1∆ cells at 18°, unlike the dosage suppression by RPO26, which promotes growth of tgs1∆ cells at 18°, albeit not as well as TGS1 or the hypomorphic CBC2 and SNP1-C∆ mutations. These findings fortify the inference that the cold sensitivity of tgs1∆ arises not from the lack of TMG caps, but from the effect of U1-bound CBC on vulnerable yeast mRNAs, among which RPO26 stands out.
Our studies shed new light on the structure-activity relations of Rpo26. We refine the margins of the minimal functional Rpo26 domain, identify essential side chains by alanine scanning, and interpret the mutational effects by reference to the crystal structure of Rpo26 in RNA polymerase II. Especially instructive were the mutations that separated the tgs1∆ dosage suppression activity of Rpo26 from its globally essential function in nuclear transcription. An appealing explanation for this separation is that certain Rpo26 mutations selectively impact Rpo26 function in one (or two) of the nuclear RNA polymerases while sparing its function in the other polymerase(s) (Tan et al. 2003). In that case, we infer that the RPO26 R135A, E124A, and R97A mutants, which are lethal or conditionally lethal with respect to rpo26∆ complementation, can provide Rpo26 function for the nuclear RNA polymerase that is most affected in tgs1∆ cells at low temperatures. That we isolated RPO31, the gene encoding the largest subunit of Pol III, in the same suppressor screen that yielded RPO26 suggests to us that Pol III is especially sensitive to the level of Rpo26 subunit in tgs1∆ cells at low temperatures. Rpo26 is in intimate contact with the large subunits of nuclear RNA polymerases during and after assembly of the polymerases (Wild and Cramer 2012) and there are well-documented genetic interactions of Rpo26 with Rpb1, the largest Pol II subunit (Archambault et al. 1990; Nouraini et al. 1996a). We speculate that the relatively weaker suppression of tgs1∆ by increased RPO31 gene dosage (compared to RPO26 suppression) reflects enhanced assembly of Pol III when Rpo26 levels are limiting.