title: A methods review on use of nonsense suppression to study 3' end formation and other aspects of tRNA biogenesis.
abstract: Suppressor tRNAs bear anticodon mutations that allow them to decode premature stop codons in metabolic marker gene mRNAs, that can be used as in vivo reporters of functional tRNA biogenesis. Here, we review key components of a suppressor tRNA system specific to Schizosaccharomyces pombe and its adaptations for use to study specific steps in tRNA biogenesis. Eukaryotic tRNA biogenesis begins with transcription initiation by RNA polymerase (pol) III. The nascent pre-tRNAs must undergo folding, 5' and 3' processing to remove the leader and trailer, nuclear export, and splicing if applicable, while multiple complex chemical modifications occur throughout the process. We review evidence that precursor-tRNA processing begins with transcription termination at the oligo(T) terminator element, which forms a 3' oligo(U) tract on the nascent RNA, a sequence-specific binding site for the RNA chaperone, La protein. The processing pathway bifurcates depending on a poorly understood property of pol III termination that determines the 3' oligo(U) length and therefore the affinity for La. We thus review the pol III termination process and the factors involved including advances using gene-specific random mutagenesis by dNTP analogs that identify key residues important for transcription termination in certain pol III subunits. The review ends with a 'technical approaches' section that includes a parts lists of suppressor-tRNA alleles, strains and plasmids, and graphic examples of its diverse uses.
authors.affiliation: Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Keshab
authors.initials: K
authors.lastname: Rijal
authors.affiliation: Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA. Electronic address: maraiar@mail.nih.gov.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Maraia
authors.affiliation: Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA. Electronic address: arimbasseriag@mail.nih.gov.
authors.forename: Aneeshkumar G
authors.initials: AG
authors.lastname: Arimbasseri
mesh_terms: D000818:Animals; D018389:Codon, Nonsense / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000379:methods; D026721:RNA 3' End Processing; D012320:RNA Polymerase III / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000096:biosynthesis*; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism*; D013489:Suppression, Genetic; D061806:Transcription Termination, Genetic
chemical_list: D018389:Codon, Nonsense; D012322:RNA Precursors; D012343:RNA, Transfer; D012320:RNA Polymerase III
keywords: RNA polymerase III; RPC11; RPC2; RPC37; RPC53; Transcription termination