Polycistronic transcripts in trypanosomes and their accumulation during heat shock: evidence for a precursor role in mRNA synthesis.

Abstract

Maturation of mRNA precursors in trypanosomes involves an apparent trans splicing event in which a 39-nucleotide miniexon sequence, common to all trypanosome mRNAs, is joined to the 5' end of a protein-coding exon. We demonstrate that the processing machinery responsible for the maturation of tubulin mRNA precursors in Trypanosoma brucei can be disrupted by heat shock. This results in an accumulation of polycistronic RNA species and a decrease in the abundance of branched splicing intermediates. At normal temperatures, tubulin polycistronic transcripts were also detected and were shown in pulse-chase experiments to be abundantly synthesized and very rapidly turned over. These results, combined with results of the heat shock experiments, suggest that these polycistronic transcripts are the precursors of the (monocistronic) tubulin mRNAs of trypanosomes.Formation of the 5' end of trypanosome mRNAs entails the addition of a 39-nucleotide miniexon or spliced-leader sequence to an mRNA precursor (reviewed in references 2 and 4). In Trypanosoma brucei this miniexon sequence initially exists as the first 39 nucleotides of an approximately 140-nucleotide capped RNA (13,45,53) termed the miniexon-derived RNA (medRNA; 6, 24, 34). Equivalent RNAs, ranging in size down to the 84-nucleotide medRNA of Crithidiafasciculata (38), have been described for a number of kinetoplastid species (34,35). In every species examined, medRNA is encoded by a segment of the nuclear genome that is tandemly repeated approximately 200 times (10,35,38,42). The function of the miniexon sequence (per se) remains unknown.Although conventional introns are apparently absent from the trypanosome genome, the involvement of splicing in the synthesis of mRNA in trypanosomes is clearly indicated by the presence of a consensus 5' splice site (36) on the medRNA immediately downstream of the miniexon sequence (6,24,34) and by consensus 3' splice sites (36) directly preceding the protein-coding exons of all genes examined (3,15,54,57,58).Several models describing the mechanism of attachment of the miniexon sequence to the acceptor pre-mRNA have been proposed (6,25,34). Recently evidence supporting a trans splicing mechanism has been provided by the discovery that the 102-nucleotide intron portion of medRNA (termed minRNA, for mini-intron RNA) can be released as a discrete product from high-molecular-weight poly(A)+ RNA by treatment with HeLa cell debranching activity (26,40,52). This suggests that medRNA participates in a trans splicing reaction in which the intron portion of medRNA becomes transiently associated with pre-mRNA through a 2'-5' phosphodiester linkage (53a Little is known about the structure of transcription units in trypanosomes. Many of the protein-coding genes of trypanosomes are found to be organized as tandem repeats (7,27,33,37,43). The protein-coding regions of all genes examined to date are continuous, lacking internal introns.The tubulin locus of T. brucei consists of approximately 15 head-to-tail repeats of alternating a and a coding se...

α‐ and β‐tubulin mRNAs of Trypanosoma cruzi originate from a single multicistronic transcript

“…This accumulation is promptly reversed when cells progress towards lag phase and when the temperature is lowered to 29°C, respectively. Similarly, Muhich and Boothroyd [32] have also shown in T. brucei that the multicistronic tubulin transcripts are also induced to accumulate by heat shock. These observations suggest that under restricted conditions of cell division (metacyclic trypomastigotes, stationary and heatshocked epimastigotes) a limited capacity for processing the putative 20.0 kb precursor RNA occurs.…”

Duplication and transcription of procyclin genes inTrypanosoma brucei

“…This might occur at the level of RNA processing (34) or by degradation of the mRNA. The regulation of stability via target sequences within the mRNA has been documented for a variety of genes ranging from ompA (35) and polynucleotide phosphorylase (36) in E. coli to the transferrin receptor (37), histone H3 (38) and the protooncogenes fos (39) and myc (40) in higher eukaryotes.…”

A complex containing CstF-64 and the SL2 snRNP connects mRNA 3′ end formation and trans-splicing in C. elegans operons

“…An interaction between polyadenylation and trans-splicing has been observed in the trypanosomes (LeBowitz et al 1993;Matthews et al 1994;Schurch et al 1994;Lopez-Estrano et al 1998), which also use 3Ј end formation and trans-splicing to process polycistronic premRNAs (Johnson et al 1987;Muhich and Boothroyd 1988;Tschudi and Ullu 1988). Interestingly, a pyrimidine-rich element was identified in the trypanosome intercistronic region that was required for both trans-splicing and 3Ј end formation (Matthews et al 1994).…”