The 2'-O-ribose methyltransferase for cap 1 of spliced leader RNA and U1 small nuclear RNA in Trypanosoma brucei

Abstract

mRNA cap 1 2'-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2'-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2'-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2'-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3'-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.

FIG. 1.

Phylogeny of a 2′-O-ribose MTase family. Shown is a phylogenetic tree of sequences calculated with the neighbor joining method and the JTT matrix (24). Values at the nodes indicate the percent bootstrap support. The main taxa are indicated.

FIG. 2.

TbMTr1 has in vitro 2′-O-ribose MTase cap 1 activity. (A) The cap 4 structure and identified enzymes. (B) α-³²P-labeled m⁷G-capped transcript was incubated with recombinant TbMT370 in the presence (+) and absence (−) of AdoMet. Following digestion with nuclease P1, resistant cap dinucleotides were resolved by TLC. Dinucleotide identities are listed at the right. ori, origin of spotting.

FIG. 3.

RNAi of TbMTr1 induces defects in SL RNA and U1 snRNA methylation and SL RNA 3′-end formation. (A) RNA collected after 9 days from pRTbMTr1 cells grown in the presence (+) and absence (−) of Tet was run on a 1.1% formaldehyde agarose gel, blotted, and probed with the TbMTr1 coding region. Ethidium bromide-stained rRNA is a loading control. (B) Schematic of the cap 4 showing possible primer extension products and interpretations of intermediate cap structures. (C) Primer extension analysis of SL RNA using total RNA from clonal pRTbMTr1 RNAi cell lines 9 days after Tet induction. Extension products were separated on a 6% polyacrylamide-8 M urea gel and run next to the cognate sequence ladder. (D) Primer extension analysis of the U1 snRNA in pRTbMTr1 RNAi samples. (E) Size analysis of SL RNA and U1 snRNA by medium-resolution acrylamide RNA blotting using oligonucleotide probes TbStloopI and TbU1-20, respectively. The number of days post-Tet addition is indicated. 5S rRNA served as a loading control.

FIG. 4.

Absence of TbMTr1 affects 5′ cap 1 and 3′ maturation. (A) Absence of TbMTr1 alleles. Shown is a Southern blot of wild-type (WT) and null (KO) clonal cell lines digested with XhoI. The products were resolved on a 1% agarose gel, visualized by ethidium bromide staining, transferred, and probed for the coding region of TbMTr1. Lane M is the 1-kb ladder (Invitrogen). (B) Altered substrate SL methylation in TbMTr1-null line. Primer extension analysis using total RNA collected from wild-type and clone 1 TbMTr1-null cell lines, as described for Fig. 3. (C) Altered cap methylation of the U1 snRNA measured by primer extension. (D) Cap 1-deficient SL RNA correlates with accumulation of 3′-extended forms. Total RNAs from samples collected from wild-type and TbMTr1-null cells were resolved through a 6% polyacrylamide-8 M urea gel and probed with a γ-³²P-labeled oligonucleotide, TbSL-40, against the SL RNA intron. Mature SL RNA is denoted by “M”.

FIG. 5.

Spliced SL has position 4 methylation despite 2′-O-ribose methylation absence at position 1. (A) Spliced SL is methylated at position 4 in TbMTr1-null lines. The poly(A)⁺-selected mRNA isolated from wild-type (WT) and TbMTr1-null (KO) cells was extended using primer TbWTexon, complementary to the exon sequence, as described for Fig. 3. In the bottom panel poly(A)⁺-selected mRNA was extended with TbSL-40 as a negative control. (B) Absence of 2′-O-ribose methylation in spliced SL. Poly(A)⁺-selected RNA was treated with tobacco acid pyrophosphatase and HK phosphatase to remove the 5′-m⁷G cap and phosphates, γ-³²P labeled by polynucleotide kinase, and digested with RNase T₂. A ladder of alkali-treated RNA (OH) for the corresponding sequence serves as a size marker. Black stars indicate common bands due to the labeling procedure; the black arrow indicates the T₂-resistant cap 4 band; the white arrow indicates the T₂-resistant band unique to TbMTr1-null cells.

FIG. 6.

TbMTr1-GFP localizes to the nucleoplasm. Clonal YTAT cell lines were used to generate a line containing a GFP gene fused in frame with the TbMTr1 gene in its endogenous locus. The expressed TbMTr1-GFP fusion protein is visualized by light microscopy under UV light illumination. The positions of the nuclear and kinetoplast DNAs are revealed by staining with DAPI. The whole cells are shown by phase contrast.