Evidence for a capping enzyme with specificity for the trypanosome spliced leader RNA

Ruan, Jiapeng; Shen, Suqin; Ullu, Elisabetta; Tschudi, Christian

doi:10.1016/j.molbiopara.2007.09.001

Cited by 22 publications

(19 citation statements)

References 47 publications

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“…Other proteins classified as essential (Alsford et al 2011) are found in the complexes, including the 117.5-kDa protein, which contains domains consistent with a role in RNA cap formation. Guanylyltransferase and N7-methyltransferase functions are required for the last two steps of eukaryotic mRNA cap formation (Ghosh and Lima 2010), however the enzyme identified here is distinct from that performing the nuclear co-transcriptional capping of the SL RNA, TbCGM1 (Ruan et al 2007;Takagi et al 2007; Sturm et al 2012). The further impact on distinct aspects of cell motility following knockdown of the TbE5 suggests unidentified roles for the complexes in the regulation of protein expression and function in these parasites.…”

Section: Discussionmentioning

confidence: 80%

“…The 47.5-kDa protein motifs are distinct, with the first containing a ferredoxin-like fold RNA-binding domain (97.7% confidence) and the second a yth domain RNA-binding motif (98.1% confidence) found in splicing factor yt521. The 117.5-kDa protein showed a superficial resemblance to the SL RNA cap 0 formation enzyme TbCGM1 (Ruan et al 2007;Takagi et al 2007) in the N7-methyltransferase domain (P-value = 0.54), as well as a bioinformatic kinship with the guanylyltransferase domain (P-value = 0.018) of a 142.1-kDa nucleoside triphosphate hydrolase/guanylyltransferase/zinc finger protein associated with the induction of stumpy bloodstream forms ( Fig. 7; Mony et al 2014).…”

Section: Knockdown Of the Tbeif4e5 Protein Affects Motilitymentioning

confidence: 99%

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eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei

Freire

Vashisht

Malvezzi

et al. 2014

RNA

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Members of the eIF4E mRNA cap-binding family are involved in translation and the modulation of transcript availability in other systems as part of a three-component complex including eIF4G and eIF4A. The kinetoplastids possess four described eIF4E and five eIF4G homologs. We have identified two new eIF4E family proteins in Trypanosoma brucei, and define distinct complexes associated with the fifth member, TbEIF4E5. The cytosolic TbEIF4E5 protein binds cap 0 in vitro. TbEIF4E5 was found in association with two of the five TbEIF4Gs. TbIF4EG1 bound TbEIF4E5, a 47.5-kDa protein with two RNA-binding domains, and either the regulatory protein 14-3-3 II or a 117.5-kDa protein with guanylyltransferase and methyltransferase domains in a potentially dynamic interaction. The TbEIF4G2/TbEIF4E5 complex was associated with a 17.9-kDa hypothetical protein and both 14-3-3 variants I and II. Knockdown of TbEIF4E5 resulted in the loss of productive cell movement, as evidenced by the inability of the cells to remain in suspension in liquid culture and the loss of social motility on semisolid plating medium, as well as a minor reduction of translation. Cells appeared lethargic, as opposed to compromised in flagellar function per se. The minimal use of transcriptional control in kinetoplastids requires these organisms to implement downstream mechanisms to regulate gene expression, and the TbEIF4E5/TbEIF4G1/117.5-kDa complex in particular may be a key player in that process. We suggest that a pathway involved in cell motility is affected, directly or indirectly, by one of the TbEIF4E5 complexes.

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Section: Discussionmentioning

confidence: 80%

Section: Knockdown Of the Tbeif4e5 Protein Affects Motilitymentioning

confidence: 99%

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei

Freire

Vashisht

Malvezzi

et al. 2014

RNA

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“…CBF5 silencing induces trans-splicing defects at the first step of splicing. Interestingly, the modification of cap 4 was perturbed in the CBF5-silenced cells (3), suggesting that SLA1 may function as a chaperone to maintain the proper secondary structure of SL RNA, essential for recognition by the methyltransferases (1,22,28,44) that mediate RNA cap 4 synthesis. SL RNA is transcribed by RNA polymerase II in a very special nuclear compartment near the nucleolus described for Trypanosoma cruzi (7).…”

mentioning

confidence: 99%

Trypanosome Spliced-Leader-Associated RNA (SLA1) Localization and Implications for Spliced-Leader RNA Biogenesis

et al. 2009

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Spliced-leader-associated RNA (SLA1) guides the pseudouridylation at position ؊12 (relative to the 5 splice site) of the spliced-leader (SL) RNA in all trypanosomatid species. Nevertheless, the exact role of this RNA is currently unknown. Here, we demonstrate that the absence of pseudouridine on Leptomonas collosoma SL RNA has only a minor effect on the ability of this RNA to function in trans splicing in vivo. To investigate the possible role of SLA1 during SL RNA biogenesis, the structure of the SL RNA was examined in permeable Trypanosoma brucei cells depleted for CBF5, the H/ACA pseudouridine synthase, lacking SLA1. Our results suggest that in the absence of SLA1, the SL RNA secondary structure is changed, as was detected by differential sensitivity to oligonucleotide-directed RNase H cleavage, suggesting that the association of SLA1 maintains the SL RNA in a structural form which is distinct from the structure of the SL RNA in the steady state. In T. brucei cells depleted for the SL RNA core protein SmD1, SL RNA first accumulates in large amounts in the nucleus and then is expelled to the cytoplasm. Here, we demonstrate by in vivo aminomethyltrimethyl UV cross-linking studies that under SmD1 depletion, SLA1 remains bound to SL RNA and escorts the SL RNA to the cytoplasm. In situ hybridization with SLA1 and SL RNA demonstrates colocalization between SLA1 and the SL RNA transcription factor tSNAP42, as well as with Sm proteins, suggesting that SLA1 associates with SL RNA early in its biogenesis. These results demonstrate that SLA1 is a unique chaperonic RNA that functions during the early biogenesis of SL RNA to maintain a structure that is most probably suitable for cap 4 modification.H/ACA RNA is a group of small nucleolar RNAs that direct pseudouridylation on rRNA and snRNAs. In almost all eukaryotes, these RNAs consist of two stem-loop structures connected by a single-stranded hinge and a tail region carrying the conserved H (AnAnnA) and ACA boxes. Two short motifs of the snoRNA base pair with the target site flanking the uridine to be isomerized (9, 25). In trypanosomes, these guide RNAs have a unique structure, composed of a single stem-loop, and carry an AGA instead of an ACA box (15, 16). All of the H/ACA molecules identified so far in trypanosomes can guide rRNA pseudouridylation; however, molecules that guide analogous modifications on U snRNAs (U1 to U5) have not been identified to date. In higher eukaryotes, the guide RNAs that direct such modifications are localized in special subnuclear domains, the Cajal bodies (6). In trypanosomes, all mRNAs are processed by trans splicing. During the trans-splicing reaction, a common spliced-leader (SL) sequence of 39 nucleotides (nt) is added to the pre-mRNA from a small RNA, the SL RNA. A novel snoRNA, termed SLA1 (SL-associated RNA), in Trypanosoma brucei was initially discovered by virtue of its efficient cross-linking with the bifunctional reagent aminomethyltrimethyl (AMT) to the SL RNA (39). It was originally proposed that SLA1 is the trypanosome U5 sn...

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“…The high level of sequence similarity indicates that these genetic neighbors arose through a duplication event in the genome. TbCE1 is not thought to perform the cap 0 addition on the SL RNA, as that task is ascribed to the triphosphatase TbCET1 (15) and the bifunctional guanylyltransferase TbCGM1 (16,17).…”

Section: Immunolocalization Of the Tbe6mentioning

confidence: 99%

“…This trio of activities is found in various combinations in different systems, including three separate proteins in yeast, a pairing of the first and second activities in metazoa and plants or the second and third activities in kinetoplastids, and a single trifunctional enzyme in several viruses (14). In kinetoplastids the proteins adding cap 0 cotranscriptionally to the SL RNA are identified as TbCET1, a triphosphatase, and bifunctional TbCGM1, a guanylyltransferase and methyltransferase (15)(16)(17). Subsequent methylation of downstream nucleotides, referred to as cap 1, cap 2, and cap 4, can enhance translation levels (18).…”

mentioning

confidence: 99%

Trypanosoma brucei Translation Initiation Factor Homolog EIF4E6 Forms a Tripartite Cytosolic Complex with EIF4G5 and a Capping Enzyme Homolog

et al. 2014

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T he operon arrangement used by prokaryotes is an elegant solution to the question of regulated gene expression, with coordinated transcription of genes encoding enzymes within a given metabolic pathway under the control of a single promoter. In contrast, the majority of eukaryotes evolved independent promoters to control the expression of individual genes, and promoter types fall into classes that are activated or repressed in synchrony with functionally linked genes. Kinetoplastids employ an unusual blend of these two strategies, the constitutive transcription of polycistronic gene clusters that, apart from tandem gene arrays, typically show no discernible biochemical linkage within arrays (1, 2). The result is the virtual absence of genetic control at the level of gene transcription for mRNAs transcribed by RNA polymerase II (3, 4). Trypanosoma brucei has circumvented this limitation for the expression of a set of virulence factors associated with the variant surface glycoproteins. This family provides the coat on the cell surface and cycles a single member over time to allow this parasite to evade the host immune system. RNA polymerase I promoters provide temporal control to this gene set (5, 6). This unusual choice of polymerase is available to trypanosomes because of the mechanism that also provides a complex mRNA cap structure to all nuclear transcripts, namely, trans splicing of the spliced leader (SL) RNA (7).The SL RNA is a small, independently transcribed molecule that is the source of the hypermethylated cap 4 structure that defines nucleus-derived mRNA in kinetoplastids (8). The cap 4 structure consists of cap 0 followed by 2=-O-methylation of the first four transcribed nucleotides and an additional three methylations on the first and fourth bases (9). The first 39 nucleotides are transferred by trans splicing to each gene transcript in a polycistronic array, which, coupled with 3= polyadenylation (10), results in a monocistronic mRNA population looking very much like that from any other eukaryote with a few extra 5= methylations. Other eukaryotes widely separated from each other in evolutionary terms use this combination of polycistronic transcription and trans splicing of their own flavor of SL (11-13).RNA cap formation requires a minimum of three enzymatic activities, a triphosphatase to remove the gamma phosphate of the primary transcript, a guanylyltransferase to attach an inverted GTP cap via a triphosphate bridge, and a methyltransferase to complete the m 7 G modification that defines cap 0 (14). This trio of activities is found in various combinations in different systems, including three separate proteins in yeast, a pairing of the first and second activities in metazoa and plants or the second and third

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Evidence for a capping enzyme with specificity for the trypanosome spliced leader RNA

Cited by 22 publications

References 47 publications

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei

Trypanosome Spliced-Leader-Associated RNA (SLA1) Localization and Implications for Spliced-Leader RNA Biogenesis

Trypanosoma brucei Translation Initiation Factor Homolog EIF4E6 Forms a Tripartite Cytosolic Complex with EIF4G5 and a Capping Enzyme Homolog

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