In vivo labelling of intermediates in the discontinuous synthesis of mRNAs in Trypanosoma brucei.

Discontinuous mRNA synthesis in trypanosomes is thought to involve a 140-nucleotide precursor, called the mini-exon-derived RNA or medRNA, which contributes its 5' 35 nucleotides to the 5' end of nascent mRNAs. We used in vivo labelling of RNA to show that medRNA has a half-life of less than 6 min, whereas putative high mol. wt intermediates containing the 3' part of the medRNA have an average half-life of less than 1 min. This eliminates priming of pre-mRNA synthesis by intact medRNA as the main mode of discontinuous mRNA synthesis. Potential intermediates of 35 and 105 nucleotides were labelled in parallel with medRNA, but their significance could not be assessed in RNA preparations containing medRNA, as they are also produced by artefactual cleavage of medRNA. We show, however, that high mol. wt RNA, free of medRNA, can release medRNA segments upon a debranching treatment. These results are consistent with a trans splicing mechanism involving short-lived forked intermediates, analogous to lariats in cis splicing systems.     

Functional characterization of a 48 kDa Trypanosoma brucei cap 2 RNA methyltransferase

Kinetoplastid mRNAs possess a unique hypermethylated cap 4 structure derived from the standard m⁷GpppN cap structure, with 2′-O methylations on the first four ribose sugars and additional base methylations on the first adenine and the fourth uracil. While the enzymes responsible for m⁷GpppN cap 0 formations has been characterized in Trypanosoma brucei, the mechanism of cap 4 methylation and the role of the hypermethylated structure remain unclear. Here, we describe the characterization of a 48 kDa T.brucei 2′-O nucleoside methyltransferase (TbCom1). Recombinant TbCom1 transfers the methyl group from S-adenosylmethionine (AdoMet) to the 2′-OH of the second nucleoside of m⁷GpppNpNp-RNA to form m⁷GpppNpNmp-RNA. TbCom1 is also capable of converting cap 1 RNA to cap 2 RNA. The methyl transfer reaction is dependent on the m⁷GpppN cap, as the enzyme does not form a stable interaction with GpppN-terminated RNA. Mutational analysis establishes that the TbCom1 and vaccinia virus VP39 methyltransferases share mechanistic similarities in AdoMet- and cap-recognition. Two aromatic residues, Tyr18 and Tyr187, may participate in base-stacking interactions with the guanine ring of the cap, as the removal of each of these aromatic side-chains abolishes cap-specific RNA-binding.     

Tubulin mRNAs of Trypanosoma brucei

The tubulin genes of Trypanosoma brucei are located in a single, tightly packed cluster of ten tandemly arranged alternating alpha and beta-genes. No tubulin genes are detected outside the clustered array. Therefore, the cluster can be assumed to be the locus of tubulin gene expression. Single bands of alpha and beta-tubulin mRNAs are observed in cultured procyclic as well as in bloodstream trypanosomes. Both alpha and beta-tubulin mRNAs have distinct 5' termini, which carry a 35-nucleotide mini-exon sequence. The 3' termini of both mRNA populations are heterogeneous.     

Characterization of a Trypanosoma brucei RNA cap (guanine N-7) methyltransferase

The m7GpppN cap structure of eukaryotic mRNA is formed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase. In trypanosomatid protozoa, the m7GpppN is further modified by seven methylation steps within the first four transcribed nucleosides to form the cap 4 structure. The RNA triphosphatase and guanylyltransferase components have been characterized in Trypanosoma brucei. Here we describe the identification and characterization of a T. brucei (guanine N-7) methyltransferase (TbCmt1). Sequence alignment of the 324-amino acid TbCmt1 with the corresponding enzymes from human (Hcm1), fungal (Abd1), and microsporidian (Ecm1) revealed the presence of conserved residues known to be essential for methyltransferase activity. Purified recombinant TbCmt1 catalyzes the transfer of a methyl group from S-adenosylmethionine to the N-7 position of the cap guanine in GpppN-terminated RNA to form the m7GpppN cap. TbCmt1 also methylates GpppG and GpppA but not GTP or dGTP. Mutational analysis of individual residues of TbCmt1 that were predicted-on the basis of the crystal structure of Ecm1--to be located at or near the active site identified six conserved residues in the putative AdoMet- or cap-binding pocket that caused significant reductions in TbCmt1 methyltransferase activity. We also report the identification of a second T. brucei RNA (guanine N-7) cap methyltransferase (named TbCgm1). The 1050-amino acid TbCgm1 consists of a C-terminal (guanine N-7) methyltransferase domain, which is homologous with TbCmt1, and an N-terminal guanylyltransferase domain, which contains signature motifs found in the nucleotidyl transferase superfamily.     

mRNAs that mature through trans-splicing in Caenorhabditis elegans have a trimethylguanosine cap at their 5'' termini.

Approximately 10% of the mRNAs in the nematode Caenorhabditis elegans mature through a trans-splicing mechanism that involves the transfer of a 22-nucleotide spliced leader to the 5' end of the pre-mRNA. The spliced leader RNA exists as a small nuclear ribonucleoprotein particle and has the trimethylguanosine cap that is characteristic of eucaryotic small nuclear RNAs. We found that the trimethylguanosine cap present on the spliced leader RNA was transferred to the pre-mRNA during the trans-splicing reaction. Thereafter, the trimethylguanosine cap was maintained on the mature mRNA. This is the first example of eucaryotic cellular mRNAs possessing a trimethylguanosine cap structure.     

The synthesis of a variant-specific antigen by Trypanosoma brucei in vitro.

A variant-specific surface antigen from a cloned population of Trypanosoma brucei S42 has been isolated and partially characterized. [35S]L-methionine was found to be incorporated into this material by cells incubated in vitro in a chemically defined medium. Incorporation of [35S]L-methionine was inhibited by cycloheximide and puromycin at concentrations which are known to specifically inhibit protein synthesis in other systems. The rate of synthesis of the variant-specific antigen in vitro has been estimated to be about 8% of the rate in vivo. Newly synthesized [35S]L-methionine-labelled variant-specific antigen was incorporated into the surface coat.     

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.     

Trypanosoma brucei encodes a bifunctional capping enzyme essential for cap 4 formation on the spliced leader RNA

The 5' end of kinetoplastid mRNA possesses a hypermethylated cap 4 structure, which is derived from standard m7GpppN (cap 0) with additional methylations at seven sites within the first four nucleosides on the spliced leader RNA. In addition to TbCe1 guanylyltransferase and TbCmt1 (guanine N-7) methyltransferase, Trypanosoma brucei encodes a second cap 0 forming enzyme. TbCgm1 (T. brucei cap guanylyltransferase-methyltransferase) is a novel bifunctional capping enzyme consisting of an amino-terminal guanylyltransferase domain and a carboxyl-terminal methyltransferase domain. Recombinant TbCgm1 transfers the GMP to spliced leader RNA (SL RNA) via a covalent enzyme-GMP intermediate, and methylates the guanine N-7 position of the GpppN-terminated RNA to form cap 0 structure. The two domains can function autonomously in vitro. TbCGM1 is essential for parasite growth. Silencing of TbCGM1 by RNA interference increased the abundance of uncapped SL RNA and lead to accumulation of hypomethylated SL RNA. In contrast, silencing of TbCE1 and TbCMT1 did not affect parasite growth or SL RNA capping. We conclude that TbCgm1 specifically cap SL RNA, and cap 0 is a prerequisite for subsequent methylation events leading to the formation of mature SL RNA.     

Vaccinia virus produces late mRNAs by discontinuous synthesis

We describe the unusual structure of a vaccinia virus late mRNA. In these molecules, the protein-coding sequences of a major late structural polypeptide are preceded by long leader RNAs, which in some cases are thousands of nucleotides long. These sequences map to different regions of the viral genome and in one instance are separated from the late gene by more than 100 kb of DNA. Moreover, the leader sequences map either upstream or downstream of the late gene, are transcribed from either DNA strand, and are fused to the late gene coding sequence via a poly(A) stretch. This demonstrates that vaccinia virus produces late mRNAs by tagging the protein-coding sequences onto the 3' end of other RNAs.     

Expression of procyclin mRNAs during cyclical transmission of Trypanosoma brucei

Trypanosoma brucei, the parasite causing human sleeping sickness, relies on the tsetse fly for its transmission. In the insect, EP and GPEET procyclins are the major surface glycoproteins of procyclic (midgut) forms of the parasite, with GPEET predominating in the early procyclic form and two isoforms of EP in the late procyclic form. EP procyclins were previously detected on salivary gland trypanosomes, presumably epimastigotes, by immunoelectron microscopy. However, no procyclins could be detected by mass spectrometry when parasites were isolated from infected glands. We have used qualitative and quantitative RT-PCR to analyse the procyclin mRNAs expressed by trypanosomes in the tsetse midgut and salivary glands at different time points after infection. The coding regions of the three EP isoforms (EP1, EP2 and EP3) are extremely similar, but their 3' untranslated regions contain unique sequences that make it possible to assign the cDNAs amplified by this technique. With the exception of EP2, we found that the spectrum of procyclin mRNAs expressed in the midgut mirrors the protein repertoire of early and established procyclic forms. Surprisingly, procyclin mRNAs, including that of GPEET, are present at relatively high levels in salivary gland trypanosomes, although the proteins are rarely detected by immunofluorescence. Additional experiments using transgenic trypanosomes expressing reporter genes or mutant forms of procyclin point to a mechanism of translational or post-translational control, involving the procyclin coding regions, in salivary gland trypanosomes. It is widely accepted that T. brucei always has a coat of either variant surface glycoprotein or procyclin. It has been known for many years that the epimastigote form does not have a variant surface glycoprotein coat. The finding that this life cycle stage is usually negative for procyclin as well is new, and means that the paradigm will need to be revised.     

Trypanothione-dependent synthesis of deoxyribonucleotides by Trypanosoma brucei ribonucleotide reductase

Trypanosoma brucei, the causative agent of African sleeping sickness, synthesizes deoxyribonucleotides via a classical eukaryotic class I ribonucleotide reductase. The unique thiol metabolism of trypanosomatids in which the nearly ubiquitous glutathione reductase is replaced by a trypanothione reductase prompted us to study the nature of thiols providing reducing equivalents for the parasite synthesis of DNA precursors. Here we show that the dithiol trypanothione (bis(glutathionyl)spermidine), in contrast to glutathione, is a direct reductant of T. brucei ribonucleotide reductase with a K(m) value of 2 mm. This is the first example of a natural low molecular mass thiol directly delivering reducing equivalents for ribonucleotide reduction. At submillimolar concentrations, the reaction is strongly accelerated by tryparedoxin, a 16-kDa parasite protein with a WCPPC active site motif. The K(m) value of T. brucei ribonucleotide reductase for T. brucei tryparedoxin is about 4 micrometer. The disulfide form of trypanothione is a powerful inhibitor of the tryparedoxin-mediated reaction that may represent a physiological regulation of deoxyribonucleotide synthesis by the redox state of the cell. The trypanothione/tryparedoxin system is a new system providing electrons for a class I ribonucleotide reductase, in addition to the well known thioredoxin and glutaredoxin systems described in other organisms.     

The 2'-O-ribose methyltransferase for cap 1 of spliced leader RNA and U1 small nuclear RNA in Trypanosoma brucei

mRNA cap 1 2'-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2'-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2'-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2'-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3'-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.