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A tRNA(CACVal) gene variant, pHtV4, was cloned from human placenta genomic DNA. This gene differs from a closely related, functional tRNA(CACVal) 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(CACVal) 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.