Mutations in the RNA polymerase III subunit Rpc11p that decrease RNA 3' cleavage activity increase 3'-terminal oligo(U) length and La-dependent tRNA processing.

The 3′ ends of tRNA and other RNA polymerase III (Pol III)-dependent genes contain dTn termination signals at which Pol III pauses and releases its RNA (6). The role of dTn extends beyond termination, since it provides a means to link Pol III transcripts to La, an abundant and ubiquitous nuclear phosphoprotein that binds these RNAs in a 3′-oligo(U) length-dependent manner and promotes their posttranscriptional processing (27, 32, 37). Although 3′-U length heterogeneity has been well documented for Pol III transcripts (27), relatively little is known about the mechanisms involved and its functional significance. A model system that can be used to alter 3′-U length and study its consequences should be helpful in understanding functional connectivity between Pol III termination and posttranscriptional processing. La protein protects pre-tRNAs from 3′ exonucleases and imposes order on posttranscriptional processing so that 5′ processing precedes 3′ processing for many pre-tRNAs (38). Finding the reverse order suggests that different pre-tRNAs use the La-independent and -dependent pathways to various degrees (25). While La-homologous protein (Lhp1p) is nonessential in the yeast Saccharomyces cerevisiae (and in Schizosaccharomyces pombe), its deletion causes lethality or growth deficiency in combination with mutations that impair base pairing or modification of certain pre-tRNAs, indicating that defective pre-tRNAs can be salvaged by La (5, 7, 21, 38). Decay of hypomodified occurs via 3′ adenylation and exonucleolytic degradation of in a process called nuclear surveillance (22) but can be rescued by excess Lhp1p (1, 5). An opal suppressor tRNASerUGAM gene with a dT7 terminator (tRNAmSer7T), which suppresses a nonsense mutation in the ade6-704 allele and accumulation of red pigment, has been used to study tRNA biogenesis and the function of the La homolog, Sla1p, in the yeast S. pombe. tRNASerUGAM expression is dependent on accurate and efficient initiation and termination by Pol III and on La for processing (12-14, 17, 18). Mutations in pre-tRNASerUGAM that confer dependence on La also appear to cause it to be temperature sensitive, recognized poorly by RNase P, and cleaved aberrantly (13, 35). Thus, pre-tRNASerUGAM is processed inefficiently in sla1+ cells, yielding partial suppression, as reflected by pink colony color (no suppression yields red colonies, and full suppression yields white colonies), while the pre-tRNASerUGAM does not accumulate or produce mature tRNASerUGAM or any suppression in sla1 deletion cells (18). These data suggest that although pre-tRNASerUGAM is defective and cannot survive without La, it can be salvaged by La to produce mature functional tRNASerUGAM. However, any pre-tRNASerUGAM directed to the La-independent pathway would not survive to maturation, even in sla1+ cells. Related to Pol III termination is pausing at dTn by Pol II during mRNA synthesis (10, 36). The protein TFIIS uses a zinc ribbon dipeptide, Asp-Glu, to promote 3′ cleavage of RNA stalled in the Pol II active site to resume elongation (20, 23). Rpc11p is an integral Pol III subunit, homologous to TFIIS in its Asp-Glu zinc ribbon, that mediates RNA 3′ cleavage and facilitates Pol III termination (8). Indeed, Pol III can carry out RNA 3′ cleavage and resynthesis while pausing at dTn (4), and this may help overcome kinetic barriers to termination (3, 8). We used a partial-suppression phenotype mediated by tRNASerUGAM in S. pombe strain yYH1 to screen for dominant gain-of-suppression mutants of rpc11. Pre-tRNAs in these mutants undergo remarkably efficient processing along the La-dependent pathway of tRNA maturation. The mutant Pol III enzymes exhibit reduced 3′ cleavage activity in vitro and produce RNAs with longer 3′ Us in vitro and in vivo. We also found that Sla1p prefers RNAs with at least four Us. Since a majority of pre-tRNAs isolated from wild-type (WT) yeast have ≤3 Us, this preference helps explain the magnitude of the rpc11 mutant effects. It would appear that pre-tRNAs that would normally decay because they have too few Us for efficient La binding are instead shifted to the La-dependent pathway of tRNA maturation in the mutants. Since defective pre-tRNAs are sensitive to the loss of La, the cleavage activity of Rpc11p may also contribute to nuclear surveillance (22).