Trypanosoma brucei spliced 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 (psi) 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 psi(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 psi(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.

FIG. 1.

Identification and purification of the TbMTr1 complex. (A) Western blot probing alternate fractions of a 2-to-20% sucrose gradient loaded with either purified His-tagged, E. coli-expressed TbMTr1 (rTbMTr1-His) with anti-His antibody (top) or the TbMTr1-HA fusion protein (TbMTr1^−/HA) from T. brucei nuclear extracts with monoclonal anti-HA antibody (bottom). Diamonds indicate the following protein standards: soybean trypsin inhibitor (20.1 kDa), bovine serum albumin (67 kDa), lactate dehydrogenase (140 kDa), and catalase (232 kDa). (B) Western blot using peroxidase anti-peroxidase reagent detecting the TbMTr1-PTP fusion protein in whole-cell extracts resolved with a Benchmark prestained molecular mass marker. (C) Colloidal Coomassie stain of SDS-PAGE gel from final purification step of TbMTr1-PTP purification (right lane). Bands are labeled with apparent molecular masses compared to that of the Benchmark protein ladder (left and center lanes) (Invitrogen).

FIG. 2.

SLA1 snoRNA specifically associates with TbMTrl complex. Primer extension analysis of RNA species in TbMTr1-HA immunoprecipitated samples from 3% input (I), 3% supernatant (S), and 10% pellet (P) performed with HA.11 monoclonal antibody. Oligonucleotides targeting the SLA1, Tb6Cs1H2, and Tb9Cs2H1 H/ACA snoRNAs, TbMTr1 substrates SL RNA and U1 snRNA, and the C/D box U3 snoRNA were used.

FIG. 3.

SL RNA is underprocessed in TbMTAP^−/− cells. (A) The SL RNA-modified nucleotides implicated in kinetoplastid trans splicing and translation are shown within the exon sequence. The nomenclature for cap intermediates is listed above the modified nucleotides. An m above the sequence indicates base methylation; an m below the sequence indicates 2′-O-ribose methylation. (B) SL RNA cap phenotype in the wild type (WT) and TbMTAP knockout (MTAP KO) (center) determined using γ-³²P-labeled oligonucleotide specifically targeting substrate SL RNA in primer extensions (left). Quantification of cap intermediates was performed with QuantOne software from Amersham (right) and are listed as percentages of the total. (C) Primer extension with SL RNA intron-specific oligonucleotide detecting CMC attachment to ψ₂₈ in wild-type (WT), TbMTr1 knockout (MTr1 KO), and TbMTAP knockout (MTAP KO) RNA samples. (D) Primer extension analysis of hydrazine-aniline-treated RNA samples as described above for detection of unmodified uridine nucleotides at position 28 of the SL RNA. (E) High-resolution RNA blot probed for substrate SL RNA in wild-type and TbMTAP^−/− cells with radiolabeled oligonucleotide complementary to the intron sequence. 3′ ext., 3′-extended forms. (F) Mature mRNA cap structures are undermethylated in TbMTAP-null mutants. Primer extension assay on poly(A)-purified RNA with [γ-³²P]-labeled oligonucleotide complementary to SL exon sequence for wild type (WT) and TbMTAP knockout (MTAP KO).

FIG. 4.

TbMTAP is required for SL processing complex formation. (A) Western blot probing for TbMTr1-HA fusion protein using polyclonal rabbit HA antibody in HA.11 monoclonal antibody immunoprecipitation input (I), supernatant (S), and pellet (P) from TbMTr1^−/HA and TbMTr1^−/HA TbMTAP^−/− cells. The asterisk indicates the nonspecific band detected by the polyclonal antibody. (B) Primer extension using oligonucleotide complementary to the SLA1 snoRNA from 2% input (I), 2% supernatant (S), and 10% pellet (P) RNA isolated by TbMTr1-HA immunoprecipitation from TbMTr1^−/HA and TbMTr1^−/HA TbMTAP^−/− cells. (C) Western blot with monoclonal anti-HA antibody probing alternate fractions of a 2-to-20% sucrose gradient loaded with nuclear extracts from either TbMTr1^−/HA (top) or TbMTr1^−/HA TbMTAP^−/− cells (bottom). Diamonds indicate the following protein standards: soybean trypsin inhibitor (20.1 kDa), bovine serum albumin (67 kDa), lactate dehydrogenase (140 kDa), and catalase (232 kDa).

FIG. 5.

Members of the TbMTr1 complex for SL RNA processing. Six polypeptides and the SLA1 snoRNA coordinate cap 1 2′-O-ribose methylation and pseudouridine formation on SL RNA in T. brucei. Spatial organization is speculative. *, SL RNA is not found in stable association with the complex.

FIG. 6.

Working model for SL RNA biogenesis. The enzymatic reactions required for maturation of the SL RNA are shown. Gray backgrounds indicate enzymes that have not been assigned. Assembly of the heptameric Sm ring defines early and late steps in the process. RNA synthesis (16, 26, 68) and cap 0 formation (54) occur at a discrete site in the nucleoplasm. Cap 1 modifications and ψ₂₈ formation are early modifications; however, their relative order is not known. TbNhp2 and TbNop10 localize to the nucleolus (6, 55); thus, TbMTr1 complex modifications are placed in that location. The two-step removals of the poly(U) tail and the remaining four cap methylations are late Sm-dependent events. The relative timing of 5′ and 3′ processing is not known.