The common 5'' terminal sequence on trypanosome mRNAs: a target for anti-messenger oligodeoxynucleotides.

Several mature mRNAs of Trypanosoma brucei were previously shown to have a common 5' terminal sequence of 35 nucleotides (nt) encoded by a separate mini-exon. To verify whether all trypanosome mRNAs contain this mini-exon sequence at their 5' end, we have tested oligodeoxynucleotides complementary to different parts of the 35 nt leader sequence for their ability to inhibit translation of total trypanosome mRNA. All oligomers tested inhibited translation of trypanosome mRNAs in a wheat germ extract. They had no effect on translation of Brome mosaic virus mRNA and of a trypanosome mRNA for phosphoglycerate kinase modified to remove the mini-exon sequence. Three different 12mers inhibited translation 35-60%; both the 22- and 34mer inhibited translation 95-100%. Incorporation of amino acids decreased proportionally in all protein bands detected in high resolution polyacrylamide gels. Our results show that all trypanosome mRNAs that yield a product detectable in gel contain a mini-exon sequence. We infer that most, if not all, trypanosome mRNAs contain a 5' terminal mini-exon sequence acquired by discontinuous synthesis.     

Direct analysis of the mini-exon donor RNA of Trypanosoma brucei: detection of a novel cap structure also present in messenger RNA.

The mini-exon, a short segment found at the 5' end of trypanosome mRNAs, is contributed by a small RNA, the mini-exon donor (medRNA). In vivo 32P-labeled medRNA, a set of smaller RNAs related to it, and mRNA, were purified from Trypanosoma brucei by hybrid selection and gel electrophoresis. Using RNA fingerprinting and sequencing techniques, mini-exon oligonucleotides were identified and characterized. We detected a novel 5' terminal capped oligonucleotide present in both medRNA and mRNA. This structure contained m7G and at least four modified nucleotides, not identified previously. If the T. brucei mini-exon has exactly four transcribed nucleotides upstream from its originally designated 5' end, it would begin with the sequence: m7GpppA*A*C*U*AA*CG (asterisks denote modification) and medRNA would be 140 nucleotides long, excluding the m7G residue. The mini-exon contains, and retains during its transfer to mRNA, a novel 5' terminal structure whose presence could confer unique functional attributes.     

Mini-exon derived RNA gene ofLeishmania donovani: structure,organization and expression

Mini-exon derived RNA is a small nuclear RNA of trypanosomatid protozoa such asLeishmania which donates its 5′-terminal 39 nucleotides to the 5’-ends of cellular messenger RNAs by trans-splicing. We have cloned a mini-exon derived RNA gene fromLeishmania donovani and studied its organization and expression. About 200 copies of the gene per haploid genome are organized as a tandem repeat on a single chromosome. The gene is transcribed as a 95-nucleotide RNA. The first 39 nucleotides of mini-exon derived RNA is also found at the 5′-terminus of a cellular mRNA (Β-tubulin), thus confirming its identity. Sequence analysis of the gene and its flanking regions showed that while classical RNA polymerase II promoter elements such as TATA and CAAT are absent from the 5′-upstream region, intragenic sequence motifs resembling RNA polymerase III promoter elements are present. The implications of this finding for mini-exon derived RNA expression are discussed.     

Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides.

Synthesis of mRNA in kinetoplastid protozoa involves the process of trans-splicing, in which an identical 39-41-nucleotide (depending on the species) mini-exon is placed at the 5' end of mature mRNAs. The mini-exon sequence is highly conserved among all members of the Kinetoplastida, nucleotides 1-6 being identical in the four genera so far examined. Prior to trans-splicing, the mini-exon donor RNA is capped by the addition of a (5'-5') triphosphate-linked 7-methylguanosine, followed by modification of the first four transcribed nucleotides. Partial structures have been previously deduced for this cap 4 moiety from Trypanosoma brucei and Leptomonas collosoma. We have purified enough cap 4 from T. brucei and Crithidia fasciculata to allow definitive structural analysis by combined liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. The results, together with the known mini-exon sequence, show that cap 4 in both species has the structure m7G(5')ppp(5')m6(2)AmpAmpCmpm3Ump. The presence of N6,N6,2'-O-trimethyladenosine and 3,2'-O-dimethyluridine, nucleosides previously unknown in nature, were confirmed by rigorous comparison with synthetic standards. The conservation of cap 4 between these divergent genera suggests that this structure may be common to most if not all Kinetoplastida.