Abstract:
U14 is a small nucleolar RNA required for the processing of eukaryotic rRNA precusors. The U14 genes of mouse as well as rat, hamster, human, Xenopus and trout are encoded within introns of the constitutively expressed 70-kDa-cognate-heat-shock protein gene (hsc70). We demonstrate here that U14.6 and U14.8 snRNAs, in addition to the previously characterized U14.5, are processed from their respective introns when hsc70 pre-mRNA transcripts containing these intronic snRNAs are injected into Xenopus oocyte nuclei. Identical intermediates are observed in the processing of all three mouse U14 snRNAs indicating similar processing pathways. The production of U14 snRNA processing intermediates possessing either mature 5' or 3' termini demonstrated that processing can occur at either end independent of maturation at the other terminus. Processing of U14.6 from hsc70 intron 6 is not dependent upon the base pairing of intron sequences flanking the 5' and 3' termini of the encoded U14 snRNA molecule. Therefore, excision of an intronic snRNA does not require extending the 5',3' terminal helix of U14 snRNA secondary structure into flanking intron regions as originally suggested. Microinjection of the plasmid vector containing the mouse hsc70/U14.5 snRNA coding region revealed that undetermined plasmid sequences can serve as non-specific promoters to generate spurious RNA transcripts. The processing of these transcripts and examination of the plasmid-initiated transcriptional-start sites indicated that a U14-specific promoter is not present in or around the intron-encoded U14.5 gene. These results strongly suggest that biosynthesis of mouse U14 snRNA results from an intron-processing pathway.