Processing of a phosphoglycerate kinase reporter mRNA in Trypanosoma brucei is not coupled to transcription by RNA polymerase II.
Capping of mRNAs is strictly coupled to RNA polymerase II transcription and there is evidence, mainly from metazoans, that other steps in pre-mRNA processing show a similar linkage. In trypanosomes, however, the mRNA cap is supplied by a trans spliced leader sequence. Thus pre-mRNAs transcribed by RNA Polymerase I are capped by trans splicing, and translation-competent transgenic mRNAs can be produced by RNA Polymerase I and T7 RNA polymerase so long as the primary transcript has a splice acceptor signal. We quantified the efficiency of processing of trypanosome pre-mRNAs produced from a plasmid integrated either at the tubulin locus, or in an rRNA spacer, and transcribed by RNA polymerase II, RNA polymerase I or T7 RNA polymerase. The processing efficiencies were similar for primary transcripts from the tubulin locus, produced by RNA polymerase II, and for RNA from an rRNA spacer, transcribed by RNA polymerase I. Primary transcripts produced by T7 RNA polymerase from the tubulin locus were processed almost as well. There was therefore no evidence for recruitment of the 3'-splicing apparatus by the RNA polymerase. Abundant transcripts transcribed from the rRNA locus by T7 RNA polymerase were somewhat less efficiently processed.
Stewart
Mhairi
M
Zentrum für Molekularbiologie der Universität Heidelberg, ZMBH-DKFZ Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
Haile
Simon
S
Jha
Bhaskar Anand
BA
Cristodero
Marina
M
Li
Chi-Ho
CH
Clayton
Christine
C
D010735:Phosphoglycerate Kinase / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics*; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016053:RNA, Protozoan / Q000378:metabolism*; D020040:Trans-Splicing; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000378:metabolism*
D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D010735:Phosphoglycerate Kinase; D012319:RNA Polymerase II