Complete Cap 4 Formation Is Not Required for Viability inTrypanosoma brucei

Abstract

In kinetoplastids spliced leader (SL) RNA is trans-spliced onto the 5 ends of all nuclear mRNAs, providing a universal exon with a unique cap. Mature SL contains an m 7 G cap, ribose 2-O methylations on the first four nucleotides, and base methylations on nucleotides 1 and 4 (AACU). This structure is referred to as cap 4. Mutagenized SL RNAs that exhibit reduced cap 4 are trans-spliced, but these mRNAs do not associate with polysomes, suggesting a direct role in translation for cap 4, the primary SL sequence, or both. To separate SL RNA sequence alterations from cap 4 maturation, we have examined two ribose 2-O-methyltransferases in Trypanosoma brucei. Both enzymes fall into the Rossmann fold class of methyltransferases and model into a conserved structure based on vaccinia virus homolog VP39. Knockdown of the methyltransferases individually or in combination did not affect growth rates and suggests a temporal placement in the cap 4 formation cascade: TbMT417 modifies A 2 and is not required for subsequent steps; TbMT511 methylates C 3 , without which U 4 methylations are reduced. Incomplete cap 4 maturation was reflected in substrate SL and mRNA populations. Recombinant methyltransferases bind to a methyl donor and show preference for m 7 G-capped RNAs in vitro. Both enzymes reside in the nucleoplasm. Based on the cap phenotype of substrate SL stranded in the cytosol, A 2 , C 3 , and U 4 methylations are added after nuclear reimport of Sm protein-complexed substrate SL RNA. As mature cap 4 is dispensable for translation, cap 1 modifications and/or SL sequences are implicated in ribosomal interaction.Most eukaryotic pre-mRNAs acquire a 5Ј 7-methyl guanosine (m 7 G) cap early during transcription elongation by RNA polymerase (pol) II. This m 7 G cap (cap 0) is attached to the 5Ј-most base of the mRNA by a 5Ј-5Ј triphosphate linkage that is formed by three separate enzymatic reactions (43). The cap 0 structure has been linked to several posttranscriptional processes, including mRNA stability, intracellular transport, and translation (4). More complex 5Ј-cap structures that include ribose 2Ј-O methylations of the first (cap 1) and second (cap 2) nucleotides appear in plants, animals, and viruses (17). In humans the cap 1 ribose 2Ј-O methylation occurs in the nucleus, while the cap 2 methylation occurs later in the cytoplasm (24, 27, 37). Additional cap modifications have been implicated in translational control in vertebrate cells (23, 24) and may be advantageous for translation of viral mRNAs (13).Nature's most complex 5Ј cap is the hypermethylated cap 4 structure found on substrate spliced leader (SL) RNA in the kinetoplastid protozoa, which include the pathogenic parasites Leishmania major, Trypanosoma brucei and Trypanosoma cruzi. Possible roles for the kinetoplastid cap structure include recognition by, and assembly of, trans-spliceosome components and/or recruitment of ribosomes to the mRNA. Systemic methylation inhibitors in T. brucei led to loss of SL transsplicing (35, 50), but these deleterious effects we...

Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1

“…Since trans splicing depends on the methylation status of the SL RNA cap4 structure (48,49) and since there is evidence that SL RNA cap4 formation occurs cotranscriptionally (31), it was possible that the trans-splicing block observed upon CRK9 silencing was due to inappropriate cap4 formation. To monitor cap4 methylation on the SL RNA, we employed a modified primer extension assay with a reduced concentration of deoxyribonucleotides and with unmodified MMLV reverse transcriptase, which terminates polymerization before the methylated nucleotide under these conditions (50,51). In noninduced cells, the SL RNAspecific extension products spanned six positions: cap0 to cap4, in which, as previously noted (52), a cap3-specific extension product is typically not observed because MTR3 methylates positions 3 and 4, and cap4, which can generate two extension products (Fig.…”

“…Thus, the most intriguing aspect of our work is the demonstration that cap4 formation is controlled by a single enzyme. Since the trypanosome cap4 structure is unique, essential for trans splicing (48,49), and important for translation (57), identification of the methyltransferases has been a priority and, thus far, the three 2=-O-ribose methyltransferases MTR1, MTR2 (also termed COM or MT48), and MTR3 (also termed MT57), which methylate positions 1, 2, and 3/4, respectively, have been identified (51,52,(58)(59)(60). Surprisingly, however, elimination of individual MTR genes left trypanosomes viable and did not impair trans splicing.…”

Binding Specificities and Potential Roles of Isoforms of Eukaryotic Initiation Factor 4E in Leishmania

“…If this were the case, we would expect to find this protein where cap 4 methylations take place. It was reported that the enzymes responsible for the 2Ј-O-methylations of the ribose residues of the SL RNA are nuclear (5,6,64) and that SL RNA modification and Sm assembly take place in the nucleus (10,38). LeishIF4E-3 is associated with a slowmigrating complex in sucrose gradients but not with polysomes.…”

The TbMTr1 Spliced Leader RNA Cap 1 2 ′-O-Ribose Methyltransferase from Trypanosoma brucei Acts with Substrate Specificity

“…The TbMTr2 in vitro activity, however, does not require cap 1-modified RNA as substrate (17). Loss of cap 2 modification in cells lacking TbMTr2 does not affect TbMTr3 activity, as downstream modifications are evident (14,16). The absence of TbMTr3 activity leads to the loss of cap 4 2Ј-O-ribose methylation, but this could be due to dual MTase activity on the part of the depleted enzyme, or to an unidentified TbMTr4 that requires prior substrate methylation(s).…”

“…The radiolabeled spots were then quantitated to estimate the extent of cap 1 methylation (m 7 G*pppAm/[m 7 G*pppAp ϩ m 7 G*pppAm]). For RNase T2 digestion, the radiolabeled RNA was digested in 50 mM ammonium acetate, pH 4.5, and 2 mM EDTA with 40 units/ml RNase T2 at 37°C for 12 h. Digestion products were resolved on 25% acrylamide, 8 M urea gels and visualized by PhosphorImager (14).…”