A protein related to the vaccinia virus cap-specific methyltransferase VP39 is involved in cap 4 modification in Trypanosoma brucei.

The spliced-leader (SL) RNA plays a key role in the biogenesis of mRNA in trypanosomes by providing the m(7)G-capped SL sequence to the 5' end of every mRNA. The cap structure of the SL RNA is unique in eukaryotes with 4 nucleotides after the cap carrying a total of seven methyl groups and by convention is referred to as "cap 4". Although the enzymatic machinery for cap addition has been characterized in several organisms, including Trypanosoma brucei, the identification of methyltransferases dedicated to the generation of higher order cap structures has lagged behind, except in viruses. Here we describe T. brucei MT57 (TbMT57), a primarily nuclear polypeptide with structural and functional similarities to vaccinia virus VP39, a bifunctional protein acting at the mRNA 5' end as a cap-specific 2'-O-methyltransferase. Down-regulation by RNAi or genetic ablation of TbMT57 resulted in the accumulation of SL RNA missing 2'-O-methyl groups at positions +3 and +4 and thus bearing a cap 2 rather than a cap 4. Furthermore, competitive binding studies indicated that modifications at the +3 and +4 positions are important for binding to the nuclear cap-binding complex. Genetic ablation of MT57 resulted in viable cells with no apparent defect in SL RNA trans-splicing, suggesting that MT57 is not essential or that trypanosomes have developed alternate mechanisms to counteract the absence of this protein. Interestingly, MT57 homologs are only found in trypanosomatid protozoa that have a cap 4 structure and in poxviruses, of which vaccinia virus is a prototype.

Functional characterization of a Trypanosoma brucei TATA-binding protein-related factor points to a universal regulator of transcription in trypanosomes.

Transcriptional mechanisms remain poorly understood in trypanosomatid protozoa. In particular, there is no knowledge about the function of basal transcription factors, and there is an apparent rarity of promoters for protein-coding genes transcribed by RNA polymerase (Pol) II. Here we describe a Trypanosoma brucei factor related to the TATA-binding protein (TBP). Although this TBP-related factor (TBP-related factor 4 [TRF4]) has about 31% identity to the TBP core domain, several key residues involved in TATA box binding are not conserved. Depletion of the T. brucei TRF4 (TbTRF4) by RNA interference revealed an essential role in RNA Pol I, II, and III transcription. Using chromatin immunoprecipitation, we further showed that TRF4 is recruited to the Pol I-transcribed procyclic acidic repetitive genes, Pol II-transcribed spliced leader RNA genes, and Pol III-transcribed U-snRNA and 7SL RNA genes, thus supporting a role for TbTRF4 in transcription performed by all three nuclear RNA polymerases. Finally, a search for TRF4 binding sites in the T. brucei genome led to the identification of such sites in the 3' portion of certain protein-coding genes, indicating a unique aspect of Pol II transcription in these organisms.

Role of a 300-kilodalton nuclear complex in the maturation of Trypanosoma brucei initiator methionyl-tRNA.

tRNAs are transcribed as precursors containing 5' leader and 3' extensions that are removed by a series of posttranscriptional processing reactions to yield functional mature tRNAs. Here, we examined the maturation pathway of tRNA(Met) in Trypanosoma brucei, an early divergent unicellular eukaryote. We identified an approximately 300-kDa complex located in the nucleus of T. brucei that is required for trimming the 5' leader of initiator tRNA(Met) precursors. One of the subunits of the complex (T. brucei MT40 [TbMT40]) is a putative methyltransferase and a homolog of Saccharomyces cerevisiae Gcd14, which is essential for 1-methyladenosine modification in tRNAs. Down-regulation of TbMT40 by RNA interference resulted in the accumulation of precursor initiator tRNA(Met) containing 5' extensions but processed 3' ends. In addition, immunoprecipitations with anti-La antibodies revealed initiator tRNA(Met) molecules with 5' and 3' extensions in TbMT40-silenced cells, albeit at a much lower level. Interestingly, silencing of TbMT40, as well as of TbMT53, a second subunit of the complex, led to an increase in the levels of mature elongator tRNA(Met). Taken together, our data provide a glance at the maturation of tRNAs in parasitic protozoa and suggest that at least for initiator tRNA(Met), 3' trimming precedes 5' processing.

A PCR-based method for gene deletion and protein tagging in Trypanosoma brucei.

Sequence information on the Trypanosoma brucei genome is rapidly accumulating. As a consequence, there is a need for techniques to analyze gene function systematically. Here, we describe a polymerase chain reaction (PCR)-based method for direct gene deletion and the generation of epitope-tagged fusion proteins. The approach is based on methodologies developed for Saccharomyces cerevisiae and involves PCR amplification of a reporter cassette using primers containing flanking sequences specific to the target gene. The PCR product is then transfected directly into procyclic T. brucei cells, and homologous recombinants that carry the deleted or tagged target gene are identified.

Upstream elements present in the 3'-untranslated region of collagen genes influence the processing efficiency of overlapping polyadenylation signals.

3'-Untranslated regions (UTRs) of genes often contain key regulatory elements involved in gene expression control. A high degree of evolutionary conservation in regions of the 3'-UTR suggests important, conserved elements. In particular, we are interested in those elements involved in regulation of 3' end formation. In addition to canonical sequence elements, auxiliary sequences likely play an important role in determining the polyadenylation efficiency of mammalian pre-mRNAs. We identified highly conserved sequence elements upstream of the AAUAAA in three human collagen genes, COL1A1, COL1A2, and COL2A1, and demonstrate that these upstream sequence elements (USEs) influence polyadenylation efficiency. Mutation of the USEs decreases polyadenylation efficiency both in vitro and in vivo, and inclusion of competitor oligoribonucleotides representing the USEs specifically inhibit polyadenylation. We have also shown that insertion of a USE into a weak polyadenylation signal can enhance 3' end formation. Close inspection of the COL1A2 3'-UTR reveals an unusual feature of two closely spaced, competing polyadenylation signals. Taken together, these data demonstrate that USEs are important auxiliary polyadenylation elements in mammalian genes.

Downstream sequence elements with different affinities for the hnRNP H/H' protein influence the processing efficiency of mammalian polyadenylation signals.

Auxiliary factors likely play an important role in determining the polyadenylation efficiency of mammalian pre-mRNAs. We previously identified an auxiliary factor, hnRNP H/H', which stimulates 3'-end processing through an interaction with sequences downstream of the core elements of the SV40 late polyadenylation signal. Using in vitro reconstitution assays we have demonstrated that hnRNP H/H' can stimulate processing of two additional model polyadenylation signals by binding at similar relative downstream locations but with significantly different affinities. A short tract of G residues was determined to be a common property of all three hnRNP H/H' binding sites. A survey of mammalian polyadenylation signals identified potential G-rich hnRNP H/H' binding sites at similar downstream locations in approximately 34% of these signals. All of the novel G-rich elements tested were found to bind hnRNP H/H' protein and the processing of selected signals identified in the survey was stimulated by the protein both in vivo and in vitro. Downstream G-rich tracts, therefore, are a common auxiliary element in mammalian polyadenylation signals. Sequences capable of binding hnRNP H protein with varying affinities may play a role in determining the processing efficiency of a significant number of mammalian polyadenylation signals.