An Efficiently Transcribed Human tRNA Val Gene Variant Produces a Stable Pre-tRNA: Repair of the Processing Defect by In Vitro Mutations

Abstract

A tRNA_CAC ^Val gene variant, pHtV4, was cloned from human placenta genomic DNA. This gene differs from a closely related, functional tRNA_CAC ^Val gene by four base exchanges: T residues in place of C₂₅, C₆₂, and C₆₆ create G:U pairs, and an A instead of G₆₅ creates an A:C mismatch in the corresponding RNA transcript. The tRNA^Val gene variant in pHtV4 is efficiently transcribed in HeLa cell nuclear extracts; however, the resulting pre-tRNA is processing-deficient, i.e., neither its 5′- nor its 3′-flanking sequences are removed to generate mature tRNA. Reversion of all four point mutations in pHtV4 by oligonucleotide-directed mutagenesis yielded a functional tRNA_CAC ^Val gene within the flanks of pHtV4, the pre-tRNA of which was processed to mature tRNA. Construction of a chimeric tRNA^Val gene and site-directed mutagenesis of the tRNA^Val gene in pHtV4, respectively, followed by transcription and processing studies showed that each of the four mutations contributes to the processing defect of the pHtV4-derived pre-tRNA. Moreover, this revealed that G:U pairs, which are common in all tRNAs, can impair pre-tRNA processing and therefore do not occur in certain positions in eukaryotic tRNAs.