A common pyrimidine-rich motif governs trans-splicing and polyadenylation of tubulin polycistronic pre-mRNA in trypanosomes.
In trypanosomes, the generation of monocistronic mRNAs from polycistronic precursors is achieved via RNA processing, namely trans-splicing of the spliced leader sequence at the 5' end and cleavage/polyadenylation at the 3' end of the mRNA coding region. Recent evidence raised the intriguing possibility that these two reactions are coupled. To begin a dissection of the signals required for mRNA 5'-end and 3'-end formation and to uncover potential interactions between trans-splicing and polyadenylation, we mutagenized the intergenic region between the beta- and alpha-tubulin genes of Trypanosoma brucei. Block substitutions identified the pyrimidine-rich sequences at the alpha-tubulin 3'-splice-acceptor site as a major determinant for accurate trans-splicing downstream and 3'-end formation upstream. In addition to the utilization of cryptic 3'-splice sites, obliteration of the polypyrimidine tracts led to aberrant poly(A)+ site choice, even in the presence of the wild-type poly(A)+ site and neighboring sequences. Taken together, these results indicate that the polypyrimidine-rich sequences act as a bifunctional element that affects RNA processing both upstream and downstream from itself. This is consistent with the possibility that the polypyrimidine tract is recognized by both the trans-splicing and polyadenylation machineries, either sequentially or simultaneously.
Matthews
K R
KR
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8022.
Tschudi
C
C
Ullu
E
E
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D017125:Genes, Protozoan; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D016833:Trypanosoma brucei rhodesiense / Q000235:genetics* / Q000378:metabolism*; D014404:Tubulin / Q000235:genetics*
D016054:DNA, Protozoan; D012322:RNA Precursors; D012333:RNA, Messenger; D016053:RNA, Protozoan; D014404:Tubulin; D011061:Poly A; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase