A protein related to the vaccinia virus cap-specific methyltransferase VP39 is involved in cap 4 modification in <i>Trypanosoma brucei</i>

Arhin, George K.; Li, Hongjie; Ullu, Elisabetta; Tschudi, Christian

doi:10.1261/rna.2223406

Cited by 26 publications

(43 citation statements)

References 33 publications

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“…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 were likely downstream of cap 4 formation, as previous data from our laboratory and others have demonstrated that mutations in the SL RNA, which resulted in a spectrum of cap 4 defects, can be trans-spliced in vivo (2,33,47,56). Base substitutions in the cap 4 nucleotides affected both modification of cap 4 and transsplicing in Leptomonas collosoma (34).…”

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confidence: 70%

Complete Cap 4 Formation Is Not Required for Viability inTrypanosoma brucei

et al. 2006

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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...

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confidence: 70%

Complete Cap 4 Formation Is Not Required for Viability inTrypanosoma brucei

et al. 2006

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“…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.…”

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confidence: 99%

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

Badjatia

Ambrósio

Lee

et al. 2013

Molecular and Cellular Biology

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, RPB1, mediates the enzyme's promoter escape and binding of RNA-processing factors, such as the m 7 G capping enzymes. The first critical step, Ser 5 phosphorylation, is carried out by cyclin-dependent kinase 7 (CDK7), a subunit of the basal transcription factor TFIIH. Many early-diverged protists, such as the lethal human parasite Trypanosoma brucei, however, lack the heptad repeats and, apparently, a CDK7 ortholog. Accordingly, characterization of trypanosome TFIIH did not identify a kinase component. The T. brucei CTD, however, is phosphorylated and essential for transcription. Here we show that silencing the expression of T. brucei cdc2-related kinase 9 (CRK9) leads to a loss of RPB1 phosphorylation. Surprisingly, this event did not impair RNA Pol II transcription or cotranscriptional m 7 G capping. Instead, we observed that CRK9 silencing led to a block of spliced leader (SL) trans splicing, an essential step in trypanosome mRNA maturation, that was caused by hypomethylation of the SL RNA's unique cap4.

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2006

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The 5 cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m 7 GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m 7 GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3 untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m 7 GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.Trypanosomatids are ancient eukaryotes that cycle between invertebrate vectors and mammalian hosts, causing a wide range of diseases. Leishmania parasites exist as extracellular flagellated promastigotes in the alimentary canal of female flies, and upon transfer to the mammalian host, they enter macrophages and cells of the immune system, transforming into amastigotes. Leishmania parasites are exposed to a broad range of environmental conditions, and stage differentiation is triggered by changes in temperature and pH (22,57).Trypanosomatids are characterized by a variety of unique molecular features, including polycistronic transcription of protein-coding genes (48, 60) and trans splicing (37), whereby a small leader RNA of 39 nucleotides, denoted spliced leader RNA (SL RNA), is spliced onto the 5Ј ends of all mRNAs, providing the cap structure. The trypanosomatid cap is a highly modified structure that, in addition to m 7

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

Cited by 26 publications

References 33 publications

Complete Cap 4 Formation Is Not Required for Viability inTrypanosoma brucei

Complete Cap 4 Formation Is Not Required for Viability inTrypanosoma brucei

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

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

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