Trypanosoma bruceiSpliced Leader RNA Maturation by the Cap 1 2′-O-Ribose Methyltransferase and SLA1 H/ACA snoRNA Pseudouridine Synthase Complex

Abstract

Kinetoplastid flagellates attach a 39-nucleotide spliced leader (SL) upstream of protein-coding regions in polycistronic RNA precursors through trans splicing. SL modifications include cap 2-O-ribose methylation of the first four nucleotides and pseudouridine () formation at uracil 28. In Trypanosoma brucei, TbMTr1 performs 2-O-ribose methylation of the first transcribed nucleotide, or cap 1. We report the characterization of an SL RNA processing complex with TbMTr1 and the SLA1 H/ACA small nucleolar ribonucleoprotein (snoRNP) particle that guides SL 28 formation. TbMTr1 is in a high-molecular-weight complex containing the four conserved core proteins of H/ACA snoRNPs, a kinetoplastid-specific protein designated methyltransferase-associated protein (TbMTAP), and the SLA1 snoRNA. TbMTAP-null lines are viable but have decreased SL RNA processing efficiency in cap methylation, 3-end maturation, and 28 formation. TbMTAP is required for association between TbMTr1 and the SLA1 snoRNP but does not affect U1 small nuclear RNA methylation. A complex methylation profile in the mRNA population of TbMTAP-null lines indicates an additional effect on cap 4 methylations. The TbMTr1 complex specializes the SLA1 H/ACA snoRNP for efficient processing of multiple modifications on the SL RNA substrate.Mature RNA molecules contain extensive posttranscriptional nucleotide modifications of both base and ribose moieties. The isomerization of uracil to pseudouridine (5-ribosyluracil) and 2Ј-O-ribose methylation occurs by either site-specific (31, 50) or small nucleolar RNA (snoRNA)-guided enzymes (13). Conserved snoRNA-guided rRNA nucleotide modifications occur in functionally relevant regions (14), with their absence resulting in disease states due to defective catalytic RNA activity (76). Cap ribose methylations of RNA polymerase II transcripts by capspecific methyltransferases (MTases) are conserved in higher eukaryotes and implicated in the enhancement of translational efficiency (28a).Kinetoplastid protozoa, including human-pathogenic Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi, transcribe all protein-encoding genes polycistronically (28, 40). Maturation to translatable monocistronic units requires resolution of each coding region by trans splicing of a 39-nucleotide (nt) spliced leader (SL) exon and 3Ј-end polyadenylation. The SL RNA is involved in the maturation of each and every nuclear mRNA, accounting for approximately 7% of total RNA synthesis (8, 23). Rapid substrate SL consumption supports the argument for a dynamic processing mechanism. Substrate SL RNA is modified by eight methylations of the 5-nt cap structure and pseudouridylation at nt 28 ( 28 ). Along with those of the m 7 G (cap 0), the methylations of the kinetoplastid cap 4 are the most extensive, with 2Ј-O-ribose methylation of the first four nucleotides and additional base methylations on the first (m 2 6 A) and fourth (m 3 U) positions (5, 21, 49). The SL cap 4 and/or exon primary sequence and pseudouridylation have been implicated in kinetoplast...

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

“…To support this notion, we evaluated pseudouridylation of SL RNA at position 28, which is carried out by the SLA1 ribonucleoprotein particle (RNP) that comprises several proteins, including the pseudouridine synthase CBF5 (54) and MTR1 (55). We hypothesized that if MTR1 is not recruited to the SL RNA cap in CRK9-silenced cells, then pseudouridylation may also be impaired, because MTR1 and CBF5 belong to the same RNA-protein complex.…”

“…However, these arguments are not conclusive, and there are other findings that are more in line with cotranscriptional cap4 formation. First, the interaction between SLA1 and SL RNA appears to be weak and transient, since purification of the SLA1 RNP did not copurify detectable amounts of SL RNA, indicating that base pairing alone may not be enough for SLA1 recruitment to the SL RNA (55). Second, SLA1 colocalizes with the SLRNA transcription factor SNAP2, also known as tSNAP42, suggesting that SLRNA transcription and cap1 formation occur at the same distinct site in the nucleus (32).…”

“…T. brucei MTR1 was found to be in a complex with the SLA1 RNP, which contains several subunits, including the pseudouridine synthase CBF5, and which guides pseudouridylation of SL RNA position 28 (55). While SL RNA pseudouridylation is not essential for trypanosome viability (32), CBF5 silencing affected cap4 formation, indicating that the SLA1 RNP is important for MTR1 function (54).…”

“…While SL RNA pseudouridylation is not essential for trypanosome viability (32), CBF5 silencing affected cap4 formation, indicating that the SLA1 RNP is important for MTR1 function (54). Since SLA1 can base pair with the SL RNA around the pseudouridylation site, this interaction could bring MTR1 to the SL RNA 5= end independently of RNA Pol II (55). Accordingly, we found that pseudouridylation was not impaired during CRK9 silencing, which indicates that the SLA1 RNP can interact with the SL RNA independent of phosphorylated RNA Pol II.…”

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

“…The TCA precipitated proteins were digested by trypsin and subjected to mass spectrometry as described (Zamudio et al 2009). The proteomic data were analyzed using the SEQUEST and DTASelect2 algorithms against the T. brucei genome database (Berriman et al 2005), filtering by a peptide-level false-positive rate of 5%, and a minimum of two peptides per protein (Peng et al 2002).…”