Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans splicing.
We present evidence that addition of the 35 nucleotide spliced leader (SL) to the 5' end of T. brucei mRNAs occurs via trans RNA splicing. A 100 nucleotide fragment of the 135 base SL RNA (100-mer) is revealed by S1 nuclease analysis of total and poly(A)+ RNA. This 100-mer is not detected by Northern hybridization analysis, indicating that it does not exist free in the cell. The 5' end of the 100-mer maps precisely to the conserved splice junction sequence of the SL RNA. Purified debranching enzyme releases this 100-mer RNA as a free, 100 nucleotide species. This indicates that the 100-mer is covalently linked to poly(A)+ RNA by a 2'-5' phosphodiester bond, that the branched intermediate has a discontinuous intron or Y structure (rather than a lariat), which is expected of a trans-spliced mRNA, and that the SL RNA is indeed the donor of the SL sequence to trypanosome mRNAs.
D000818:Animals; D008970:Molecular Weight; D009694:Nucleic Acid Precursors / Q000235:genetics*; D011061:Poly A / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D014158:Transcription, Genetic; D014345:Trypanosoma / Q000235:genetics*
D009694:Nucleic Acid Precursors; D012333:RNA, Messenger; D011061:Poly A