Date of Award
Doctor of Philosophy (PhD)
Cross George Laboratory
The protozoan parasite, Trypanosoma brucei, and other members of the phylogenetic order, Kinetoplastida, synthesize mRNA by a novel pathway that joins RNAs from physically unlinked gene segments. This results in the presence of a short non-coding RNA leader, called the miniexon, at the 5' end of most, probably all, mRNA. Indirect evidence suggests that trans-splicing, rather than RNA priming of transcription, is the mechanism by which the mini-exon is joined to RNA containing protein-coding sequences. In vivo 32P-labeled RNA was purified by hybrid selection with mini-exon genomic DNA, in order to analyze the structure of medRNA, the 140- nucleotide precursor of the mini-exon in mRNA. Some possible intermediates in the joining reaction and the 5' mini-exon cap structure were detected and characterized. Using this direct RNA analysis, the 51 and 31 ends of medRNA were precisely mapped and an internal modification was detected. Two small mini-exon-selected RNAs, a 39mer and a 37mer, were purified, sequenced and found to consist of the free mini-exon and a 31 - truncated version, respectively. Both ended with a 3' OH. The 39mer is a candidate biosynthetic intermediate because its structure is consistent with the proposed trans-splicing mechanism of joining. The cap structure from T. brucei mRNA was found to consist of a novel "cap 4" structure: m7GpppA*A*C(2'-O-m)U*A, where asterisks denote modification. The modified nucleotides in the cap were sequenced by a combination of partial and complete digests with non-specific nucleases. The four encoded, modified nucleotides are likely to have 2'-O-methylations, and possibly two other unidentified base modifications. The mRNA cap was indistinguishable from the medRNA cap. The cap structure is likely to be similar in the other Kinetoplastidae. This hypermethylated cap structure may have a functional role in discontinuous mRNA synthesis. These findings will be useful in future in vitro and in vivo studies. In particular, an authentic trypanosome cap may be necessary to obtain an in vitro mini-exon joining reaction.
Freistadt, Marion Struik, "The mRNA Cap and Discontinuous Transcription in Trypansoma brucei" (1988). Student Theses and Dissertations. 384.