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

Abstract

Termination by RNA polymerase III (Pol III) produces RNAs whose 3′ oligo(U) termini are bound by La protein, a chaperone that protects RNAs from 3′ exonucleases and promotes their maturation. Multiple reports indicate that yeasts use La-dependent and -independent pathways for tRNA maturation, with defective pre-tRNAs being most sensitive to decay and most dependent on La for maturation and function. The Rpc11p subunit of Pol III shows homology with the zinc ribbon of TFIIS and is known to mediate RNA 3′ cleavage and to be important for termination. We used a La-dependent opal suppressor, tRNA^SerUGAM, which suppresses ade6-704 and the accumulation of red pigment, to screen Schizosaccaromyces pombe for rpc11 mutants that increase tRNA-mediated suppression. Analyses of two zinc ribbon mutants indicate that they are deficient in Pol III RNA 3′ cleavage activity and produce pre-tRNA^SerUGAM transcripts with elongated 3′-oligo(U) tracts that are better substrates for La. A substantial fraction of pre-tRNA^SerUGAM contains too few 3′ Us for efficient La binding and appears to decay in wild-type cells but has elongated oligo(U) tracts and matures along the La-dependent pathway in the mutants. The data indicate that Rpc11p limits RNA 3′-U length and that this significantly restricts pre-tRNAs to a La-independent pathway of maturation in fission yeast.

ACKNOWLEDGMENTS

We thank M. Blum for medium preparation, M. Kawano for advice on RNA ligation, M. Cashel for advice on mutagenesis and yeast growth in liquid, H. Levin for the plasmid rescue protocol, K. Joshipura for plasmid rescue, and T. Kokubo for anti-Ptr6p. We are deeply grateful to M. Kashlev and M. Kireeva for teaching us the cleavage assay and for advice and discussion. We thank C. Kane for critical reading; A. Hinnebusch, The Friday Seminar, and SMCB members for discussion; and reviewers for comments.

R.J.M. receives support from the Commissioned Corps, USPHS. A.M. and S.H. were supported by the NICHD postbaccalaureate program.