tRNA 2'-O-methylation by a duo of TRM7/FTSJ1 proteins modulates small RNA silencing in Drosophila

Abstract

2'-O-Methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while CG5220 methylates position C32 in the same tRNAs and also targets additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as lifespan reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalyzing Nm at specific tRNAs and small RNA-induced gene silencing pathways.

Figure 1.

Identification of CG7009 and CG5220 as conserved TRM7 family proteins. (A) The automiG sensor. automiG carries a copper-inducible promoter (PMT) that drives the expression of two miRNAs (miG1 and miG2) and the GFP mRNA. Both miRNAs target the GFP mRNA with perfect complementarity. AutomiG repression is dependent on Ago2, Drosha, Pasha, Dicer-1 and Dicer-2 functions as reported previously (66). (B) CG7009 function affects automiG repression. Cells were soaked with the indicated double-stranded RNA (dsRNA), followed by automiG induction using copper sulfate. GFP expression was analyzed by western blotting. The Mbf1 protein was used as loading control. dsRNA against Fushi tarazu (Ftz) and luciferase (luc) served as negative KD controls. kDa: kilo Dalton. (C) Multiple amino acid sequence alignment of CG7009, CG5220, TRM7 (S. cerevisiae), and FTSJ1. The conserved predicted catalytic tetrad amino acids K–D–K–E are marked by red asterisks. Dark blue points to conserved amino acid in the three organisms. (D) Drosophila species evolved two TRM7 family proteins. Phylogenetic analysis of TRM7 and SBP1 MTases. The SBP1 family member RrmJ acting on rRNA was used as an outgroup. Color blue indicates TRM7 family proteins in Drosophila species other than D. melanogaster. Red indicates TRM7 family proteins in D. melanogaster.

Figure 2.

CG7009 and CG5220 affect small RNA silencing pathways. (A) Homozygous CG7009, CG5220 double mutant flies display reduced adult weight and size. Images of adult females and males CG7009, CG5220 homozygous double mutants (homo) compared to heterozygous double mutants (hetero). Below the images the average weight for flies in milligrams (mg) calculated for 3 days-old flies measured on a precision balance (n > 100 flies/ genotype; P-value < 0.001 in a Student's t-test) is indicated. The percentage change for female heterozygous CG7009 and CG5220 mutants versus homozygous CG7009, CG5220 double mutant represents a decrease of 20.5%. The percentage change for male heterozygous CG7009, CG5220 double mutant versus homozygous CG7009, CG5220 double mutant represents a decrease of 8%. (B) CG7009 and CG5220 modulate Ago2-dependent gene silencing in somatic tissues. The UAS>automiW construct is a sensor derived from automiG by which two miRNAs target the white gene (68). KD indicates eye-specific GMR-Gal4/UAS-RNAi-mediated inactivation of the respective genes (white, CG7009, CG5220 or Ago2). Canton-S was used as control for eye color determination. Darker eye coloration than Canton-S (top right) indicates that the white gene is not inactivated by Ago2-loaded miRNAs targeting white. Images were taken at the same age (5 days after hatching) for different genotypes. (C) The siRNA-dependent viral defence is compromised in CG7009 and CG5220 mutants. RT-qPCR using Drosophila C Virus (DCV)-specific primers three days after injection with DCV solution or solution free of DCV as control (not shown) in heterozygous CG7009^e02001 mutants (Control) or homozygous CG7009^e02001 (Mut CG7009) and CG5220^K>A (Mut CG5220) mutants. Shown is DCV expression relative to Rp49. Error bars represent the standard deviation (s.d.) of the mean between two (n = 2) biological replicates (n is a mix of 2–3 flies). (D) Endogenous siRNA (esi-2.1) expression is reduced in CG7009 and CG5220 mutants. Northern blotting on total RNAs extracted from adult flies of the indicated genotypes was performed using esi-2.1, bantam-specific probes and a 5S rRNA probe as loading and transfer control. nt: nucleotide. (E) The CG7009 mutation is associated with ovarian size reduction. The images show representative examples of ovaries from 4 days-old fertilized females raised on fresh yeast from trans-heterozygous CG7009^e02001/Def9487 mutants (Mut CG7009) and Rescue CG7009 (BAC)/ +; CG7009^e02001/Def9487 mutants (Rescue CG7009); n ≥ 10 for each genotype; Mut: mutant. An unpaired Mann–Whitney (Wilcoxon) test was used to calculate the significance of the ovary area differences between mutant and rescue genotypes. The percentage change from the mutant and the rescue genotypes represents a decrease of 10.5% with a P-value < 0.05 (W = 23, P-value = 0.02416). (F)CG7009 and CG5220 are involved in gypsy TE-repression in Drosophila ovaries. The Gypsy::LacZ sensor is silenced through tj>Gal4-mediated expression of an UAS-RNAi line (KD) against the white gene in follicle cells (R; tj>Gal4/ +; Gypsy::LacZ/UAS-white-RNAi). Gypsy silencing is disrupted using RNAi lines against Piwi (KD piwi), CG7009 (KD CG7009), CG5220 (KD CG5220) and CG33172 (KD CG33172). The Gypsy::LacZ sensor was also de-repressed in CG7009 null mutants (KO CG7009) and CG5220^K>A homozygous mutants (CG5220 K>A). KD: knock down; KO: knock out; no blue coloration = no β-Gal staining.

Figure 3.

Small non-coding RNA biogenesis is not globally affected in CG7009 mutants. (A) Class distribution of ovarian small RNAs (19–29 nt) matching the whole Drosophila genome reveals a significant decrease of miRNAs between control (Mut_CG7009/ +) and CG7009^e02001 homozygous mutants (Mut_CG7009). Circle circumference represents the depth of the library (indicated in million reads at the bottom right). n = 2 libraries for each genotype: Mut_CG7009: CG7009^e02001 homozygous mutant, while CG7009^e02001/ + represents the control heterozygous condition. Color code in the middle panel indicates each small RNA read matching to a category of Drosophila small RNA (miRNA: microRNA derived sequences, rRNA: rRNA derived sequences, snoRNA: small nucleolar RNA, gene indicated small RNA sequences derived from a coding genes, etc.). Sequencing of two CG7009^e02001 homozygous mutant libraries (M = 201490, SD = 21875.26) showed significantly decreased miRNA read numbers t(2) = 5.89735 when compared to the two CG7009^e02001/+ control libraries (M = 307354, SD = 14248.2). The P-value is 0.04867. The result was significant at P < 0.05. (B) Size distribution (19–29 nt) of small RNA read counts matching TE-derived sequences in Drosophila ovaries. One experiment is shown for each genotype, Mut_CG7009: CG7009^e02001 homozygous mutant, while CG7009^e02001/ + represents the control heterozygous condition. Horizontal grey line indicates the highest value and is depicted for better comparison between the two presented conditions. (C) Size distribution of small RNA read counts from ovaries matching gypsy retro-element sequences reveals that 23–29 nt piRNAs against gypsy are reduced in CG7009 mutants compared to controls. Positive and negative values correspond to sense (red) and antisense (blue) reads, respectively. Horizontal grey lines indicate the highest values (sense and antisense) and are depicted for better comparison between the two presented conditions.

Figure 4.

Mutations in CG7009 and CG5220 affect lifespan and mobility. (A) Simultaneous downregulation of CG7009, CG5220 expression results in reduced lifespan. Survival curves of males expressing an RU486-inducible RNAi transgene against CG7009 and CG5220 with (RU200) or without (RU0) RU486-mediated RNAi transgene induction. Constitutive expression (RU200) of CG5220, CG7009 KD transgenes was induced by RU486 exposure (20 mg/ml during adulthood). The curves represent the average values of five biological replicates of 30 flies per experiment. (B) Homozygous double mutant CG5220^K>A, CG7009^e02001 results in reduced lifespan. Survival curves of indicated males homozygous mutant for CG5220^K>A(Mut CG5220), homozygous mutant for CG7009^e02001 (Mut CG7009), homozygous double mutant CG5220^K>A, CG7009^e02001 (Double Mut) and heterozygous CG7009^e02001/+ used as control condition (Control). The curves represent the average values of five biological replicates of 30 flies per experiment. (C) CG7009 and CG5220 control fly behavior. Bar graphs represent data of 16 days-old male or female flies (10 flies/experiment) that climbed over 10 cm in 10 s (≥6 independent measurements for each genotype) and the standard deviation of the mean. *P < 0.01; ***P < 0.0001 in a Student's t-test.

Figure 5.

CG7009 & CG5220 are TRM7-like tRNA Nm MTases in Drosophila. (A) Presence (present) or absence (absent) of Nm-containing RNA fragments and their sizes (m/z in Daltons) upon RNase A or T1 digestion of tRNA^Phe(GAA) extracted from Drosophila adult heterozygous CG7009^e02001 mutants (Control), homozygous CG7009^e02001 mutants (CG7009 e02001), homozygous CG7009 mutants rescue (CG7009 BAC Rescue), homozygous CG5220^K>A mutants (CG5220 K>A), and double homozygous mutants CG7009^e02001,CG5220^K>A (Double mutant). (B) MALDI TOF-MS spectrum of fragments resulting from RNase T1 digestion of tRNA^Phe-(GAA) originating from heterozygous adult CG7009^e02001/+ mutants (Control), homozygous CG7009^e02001 mutants (Mutant CG7009^e02001), red line indicates the maximum value (1318 Da) and is depicted for better comparison between the 2 peaks values. Homozygous CG5220^K>A mutants (Mutant CG5220^K>A) and double homozygous mutants CG7009^e02001,CG5220^K>A (Double Mutant CG7009^e02001,CG5220^K>A) as indicated. Relevant peaks are identified by their m/z values in Daltons (X-axis). (C) RiboMethSeq analysis of Nm at positions C₃₂ and G₃₄ in tRNA^Phe(GAA) from whole heterozygous adult CG7009^e02001/+ mutants (Control), homozygous CG7009^e02001 mutants (CG7009^e02001) and rescued CG7009^e02001 mutants (Rescue CG7009 (BAC)) as indicated. Normalized cleavage efficiencies, calculated from combined 5′- and 3′-end coverage of tRNAs are shown for the neighboring nucleotides (+/– 5) of the respective ribose methylation position. The positions of interest (C₃₂ and G₃₄) in tRNA^Phe(GAA) are indicated by red arrows. Protection against cleavage is indicated (+): protected and as (–): not protected. Protection at Cm₃₂ in control flies was only moderate, indicating incomplete tRNA methylation (+low). (D) Methylation scores (MethScore) for five 2′-O-methylated positions in tRNAs showing altered methylation in CG5220 and/or CG7009 indicated mutants. MethScore (Score C), representing the level of ribose methylation was calculated from protection profiles. Data are shown for positions 32 and 34 in different D. melanogaster tRNAs as measured in heterozygous adult CG7009^e02001/+ mutants (control), homozygous CG5220^K>A mutant (mutant CG5220^K>A), two independent genetic background mutants for CG7009 (homozygous CG7009^e02001 or trans-heterozygous CG7009^e02001/Def3340 mutant), double homozygous CG7009^e02001,CG5220^K>A mutant and rescue BAC CG7009^e02001/Def3340 flies (Rescue CG7009 (BAC)). Score at Cm₃₂ for tRNA^Phe(GAA) in control flies is only moderate (not shown and Figure 5C), indicating incomplete tRNA methylation.

Figure 6.

CG33172 is a partner of the Nm-MTase complex in Drosophila. (A) Percentage of amino acid (aa) identity between CG15618, human THADA and yeast TRM732, and between CG33172, human WDR6 and yeast TRM734 (RTT10). Alignment was performed using BLAST/ BLAT tool at www.ensembl.org. (B) CG33172 and CG15618 modulate Ago2-dependent silencing in adults flies. CG33172 and CG15618 expression was knocked down by using UAS-RNAi lines and eye- specific GMR-Gal4, [w-] driver (indicated as KD), as in Figure 2B. Canton-S (wild type, [w+]) and Ago2 KD were used as controls. A darker eye coloration than Canton-expressing automiW lines (top right) indicates that the miRNAs of the sensor are failing to inactivate the white gene through impaired miRNA biogenesis or/and Ago2-dependent silencing. (C)CG33172 interacts with CG7009 in vitro. Co-immunoprecipitation of recombinant and epitope-tagged CG7009 and CG33172 after co-expression in bacteria. Western blotting using anti-GST antibody on protein extracts from input fractions co-expressing GST::CG7009 and FLAG::CG33172 and after FLAG-IP; Lower panel, Anti-GST WB reveals a GST ‘alone’ signal in the co-expressed GST and FLAG::CG33172. Inputs correspond to 10% of 10 μg of protein eluates. The expected protein sizes are 26 kDa (GST) and 62 kDa (GST::CG7009). WB, western blot; kDa, kilodaltons.

Figure 7.

Nm limits endonucleolytic cleavage of tRNA^Phe. (A) Northern blot characterization of 5′- tRNA^Phe(GAA)-derived tRFs. Northern blot on total RNAs extracted from whole heterozygous CG7009^e02001 control flies (CG7009^e02001/+), trans-heterozygous for CG7009^e02001/Def3340 or rescued mutants for CG7009 (rescue CG7009) as indicated using a 5′-tRNA^Phe(GAA) -specific probe and a 5S rRNA probe as loading and transfer control. Mature tRNA^Phe size is 73 nt (full length). 5′-tRNA^Phe-derived tRNA fragments (5′-tRF^Phe) were detected at ∼35 nt (halves). The same experiment was performed on heat-shocked flies (one hour at 37°C in a water bath), RNAs were extracted after 5 h at 25°C (indicated as HS, heat shock). nt: nucleotide. (B) The same experiment as in Figure 7A above was performed on heat-shocked flies (one hour at 37°C in a water bath). The same membrane as shown in Figure 2D was stripped and reprobed with a tRNA^Phe(GAA) -specific probe. Figures 7B and 2D thus contain identical images (marked with *) for bantam, 5S and esi-2.1 for a better comparison. RNAs were extracted after 5 hours of recovery at 25°C (indicated as HS, heat shock) with indicated genotypes. Double homozygous mutant is indicated as CG7009^e02001,CG5220^K>A. Homozygous mutant for CG7009 is indicated as CG7009^e02001. Homozygous mutant for CG5220 is indicated as CG5220^K>A. Trans-heterozygous mutants for CG7009 alleles are indicated as CG7009^e02001/Def3340 and CG7009^e02001/Def9487 nt: nucleotide.