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.     

Physical identification of branched intron side-products of splicing in Trypanosoma brucei.

Every mRNA in trypanosomes consists of two exons, a common 5' capped mini-exon or spliced leader and a coding-exon. All evidence suggests that the exons are joined by trans-splicing of two individual precursor RNAs, the mini-exon donor RNA or spliced leader precursor RNA (medRNA) and the pre-mRNA. We studied intermediates of the splicing reaction using denaturing two-dimensional PAGE and structurally identified a group of small (approximately 180-300 nt) non-polyadenylated, Y-shaped branched RNAs. The branched Y-shaped RNAs contain the 105 nt medRNA derived intron, joined in a 2'-5' phosphodiester bond to small heterogeneously sized RNAs. These non-polyadenylated branched Y-shaped RNA molecules are analogous to the lariat shaped introns of higher eukaryotes and presumably represent the released intron-like by-products of a trans-splicing reaction which joins the mini-exon and the major coding-exon.     

Multiple polyadenylation sites in a Drosophila tropomyosin gene are used to generate functional mRNAs.

The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.     

trans-spliceosomal U6 RNAs of Crithidia fasciculata and Leptomonas seymouri: deviation from the conserved ACAGAG sequence and potential base pairing with spliced leader RNA.

U6 RNA genes from the trypanosomatids Crithidia fasciculata and Leptomonas seymouri have been isolated and sequenced. As in Trypanosoma brucei, the U6 RNA genes in both C. fasciculata and L. seymouri are arranged in close linkage with upstream tRNA genes. The U6 RNA sequences from C. fasciculata and L. seymouri deviate in five and three positions, respectively, from the published T. brucei sequence. Interestingly, both C. fasciculata U6 RNA genes carry a C-->T change at the second position of the ACAGAG hexanucleotide sequence, which is important for splicing function and has been considered phylogenetically invariable. A compensatory base change of the C. fasciculata spliced leader RNA at the highly conserved 5' splice site position +5, G-->A, suggests that an interaction between the 5' splice site region and U6 RNA recently proposed for the yeast cis-splicing system may also occur in trans splicing.     

Bodo caudatus medRNA and 5S rRNA genes: tandem arrangement and phylogenetic analyses.

The Bodo caudatus mini-exon-derived RNA gene repeat has been isolated following PCR amplification. The DNA sequence of the mini-exon fits the trypanosomatid mini-exon consensus, supporting inclusion of Bodo in this group. The B. caudatus mini-exon repeat also contains the 5S ribosomal RNA gene, an organization found in the trypanosome T. rangeli and five genera of nematodes. Phylogenetic analysis of both mini-exon-derived RNA gene and 5S gene sequences show that the free-living B. caudatus is more closely related to the monogenetic Crithidia than the digenetic Trypanosoma. Similarity between the Euglena gracilis trans-spliced leader and trypanosomatid mini-exon sequences was also noted during these comparisons.