N6-methyladenosine in poly(A) tails stabilize VSG transcripts

Abstract

RNA modifications are important regulators of gene expression¹. In Trypanosoma brucei, transcription is polycistronic and thus most regulation happens post-transcriptionally². N⁶-methyladenosine (m⁶A) has been detected in this parasite, but its function remains unknown³. Here we found that m⁶A is enriched in 342 transcripts using RNA immunoprecipitation, with an enrichment in transcripts encoding variant surface glycoproteins (VSGs). Approximately 50% of the m⁶A is located in the poly(A) tail of the actively expressed VSG transcripts. m⁶A residues are removed from the VSG poly(A) tail before deadenylation and mRNA degradation. Computational analysis revealed an association between m⁶A in the poly(A) tail and a 16-mer motif in the 3' untranslated region of VSG genes. Using genetic tools, we show that the 16-mer motif acts as a cis-acting motif that is required for inclusion of m⁶A in the poly(A) tail. Removal of this motif from the 3' untranslated region of VSG genes results in poly(A) tails lacking m⁶A, rapid deadenylation and mRNA degradation. To our knowledge, this is the first identification of an RNA modification in the poly(A) tail of any eukaryote, uncovering a post-transcriptional mechanism of gene regulation.

Extended Data Fig. 1.. Chemical structures of RNA modifications found in T. brucei.

a, 15 modifications were detected in poly(A)-enriched RNA (mRNA). b, 19 modifications were not detected in poly(A)-depleted RNA. All modifications were detected in total RNA. Structures were obtained from the database Modomics (http://genesilico.pl/modomics/).

Extended Data Fig. 2.. Detection of m⁶A in T. brucei by mass-spectrometry.

a-b, Chromatograms obtained by LC-MS/MS analysis of a N6-methyladenosine standard and three RNA samples of T. brucei bloodstream form (BSF, Panel a) or insect procyclic stage (PCF, Panel b): total RNA, RNA enriched with poly(T)-beads (i.e., poly(A)-enriched RNA) and RNA that did not bind to polyT-beads (i.e., poly(A)-depleted RNA). c, Chromatogram obtained by LC-MS/MS analysis of a N1-methyladenosine standard with the 282->150 mass transition. The m¹A peak is detected at 6.5 minutes. d, Standard curve of m⁶A. Increasing quantities of commercially synthesized m⁶A were loaded on the HPLC column and the area under the chromatogram peak was measured. e, Quantification of the m⁶A/A(%) in the mRNA in a second independent experiment. n = 5 mRNA samples. (see also Source Data of Extended Figures)

Extended Data Fig. 3.. m⁶A detection in T. brucei by immunoblotting.

a, Specificity of anti-m⁶A antibody. Oligonucleotides containing either m⁶A (positive control), unmodified adenosine or m¹A (negative controls) were manually spotted in the membrane, which was incubated with anti-m⁶A antibody. The antibody specifically recognized the oligos with m⁶A, while exhibiting low cross-reactivity to the oligos with only unmodified adenosine or containing m¹A. b, m⁶A signal intensity in the immunoblot, measured by Image J, in the whole lane containing the poly(A)-enriched RNA of bloodstream forms. c, The intensity of the ~1.8 kb band was divided by the signal intensity of the entire lane. n = 5 biological replicates. d, m⁶A immunoblotting of RNA samples from two stages of T. brucei life cycle. Samples (from left to right): total RNA (Total), Poly(A)-enriched (A+) RNA and Poly(A)-depleted (A−) RNA from mammalian BSF and insect PCF. The last lane contains total mouse liver RNA (Mouse). 2 μg of total RNA, 2 μg of poly(A)-depleted RNA and 100 ng of poly(A)-enriched RNA was loaded per lane. rRNA was detected by staining RNA with methylene blue to confirm equal loading between total and poly(A)-depleted fractions. As expected rRNA is undetectable in the poly(A)-enriched fraction. (see also Supplementary Figure S1 and Source Data of Extended Figures)

Extended Data Fig. 4.. Poly(A) tail length, m⁶A and mRNA levels during VSG turnover.

a, Levels of m⁶A (immunoblot), length of VSG poly(A) tail (RNase H – northern blot) and levels of VSG mRNA (northern blot) after transcription halt by ActD. Signals were normalized to time point 0hr. The pattern observed is consistent with Figure 2b. Two-way ANOVA with sidak correction for multiple test. ( ****P<0.0001,*P=0.0190 in mRNA vs m6A in 15 min, ***P=0.0004 in poly(A) vs m6A in 15 min, ***P=0.0001 in mRNA vs m6A in 120 min,*P=0.0136 in poly(A) vs m6A in 240 min). n = 4 biological samples for mRNA and m⁶A levels, n = 3 biological samples for poly(A) tail length. Data are mean ± s.d. (see also Source Data of Extended Figures)

Extended Data Fig. 5.. Subcellular distribution of m⁶A in bloodstream form parasites.

a, Proportion of m⁶A signal in nucleus and cytoplasm. Data are mean ± s.d. n = 4 independent experiments. b, Quantification of mean fluorescence intensity (MFI) levels of m⁶A in five independent replicates in three different conditions: untreated BSF, nuclease P1 (NP1)-treated BSF, and actinomycin D (ActD)-treated BSF. Raw MFIs were obtained, the average of the untreated BSF equalled to 100%, and all other values normalized to 100%. Data are mean ± s.e.m. c, Distribution of VSG2 mRNA in the nucleus and cytoplasm in single marker (SM) cell line (single VSG expression) and in the clones that express a second reporter VSG (6 clones of DE1 express VSG117 containing a WT 16-mer motif, 7 clones of DE2 express VSG117 containing a mutagenized 16-mer motif). VSG mRNA was quantified by FISH. Nucleus was delimited by Hoechst staining. Total signal was set as 100% and the nucleus and cytoplasm represented as percentage of total signal. Error bars represent s.d. d, Microscopic observation of nuclei purified after fractionation protocol. Nuclei were stained with Hoechst. The nuclear purification was compared with initial lysates and with the cytoplasmic fraction. Scale bars, 10μm; N = 1 independent experiment. e, Western blot of subcellular fractions (total lysate, nuclear and cytoplasmic fractions) using antibodies against a nuclear protein (histone H2A; custom rabbit polyclonal 1:5000) and a cytoplasmic protein (β-tubulin; mouse monoclonal KMX-1 1:1000). For each sample, we loaded a protein equivalent to the same amount of cells. n = 3 independent experiments. (see also Supplementary Figure S1 and Source Data of Extended Figures)

Extended Data Fig. 6.. VSG double-expressor (DE) cell lines immunoblotting.

a, Overexposure of full immunoblot shown in Figure 4c. Three independent 16-mer^WT clones and three independent 16-mer^MUT are shown (C1-C6). Note that with this exposure, most intense bands are saturated. The purpose of this high exposure is to observe the region of blot corresponding to the VSG117 transcript. No VSG117 band is observed in the 16-mer^MUT clones. It is also possible to observe a weak VSG2 band in the VSG2 single expressor lane and in the 16-mer^MUT clones, which correspond to incomplete RNase H digestion of VSG2 transcript. N = 3 independent clones for each genotype (C1-C6). b, Schematics of VSG double-expressor (DE) cell-lines DE3 and DE4. VSG8 was inserted in the active bloodstream expression site, which naturally contains VSG2 at the telomeric end. In DE3, VSG8 contains its endogenous 3’UTR with the conserved 16-mer motif (sequence in blue). In DE4, the 16-mer motif of VSG8 was scrambled (sequence in orange). c, m⁶A immunoblot of mRNA from DE3 and DE4 cell-lines, in which CAF1 was further depleted by RNAi by adding Tetracycline (Tet). RNase H digestion of VSG2 mRNA was used to resolve VSG2 and VSG8 transcripts. Two independent DE3 clones and two independent DE4 clones are shown (C1-C4), each with (+) or without (−) CAF1 downregulation. (see also Supplementary Figure S1)

Extended Data Fig. 7.. CAF1 depletion.

CAF1 transcript levels measured by RT-qPCR in CAF1 RNAi cell-line used in Figure 5, Panels c-d and e-f. CAF1 downregulation was induced by adding tetracycline (Tet) to the medium. Unpaired two tailed t-test (**** P>0.0001). Data are mean ± s.d. n = 3 independent clones.

Figure 1.. m⁶A is present in the poly(A) tail of VSG mRNA and other transcripts.

a, Overlap chromatogram of nucleoside modifications detected in mRNA mammalian BSF by LC-MS/MS. Data are ratios between peak areas. B, Enrichment of nucleoside modifications in mRNA relative to total RNA. Two-way ANOVA with sidak correction for multiple test (**** m⁶A, m^6,6A, m⁷G and m¹A P<0.0001). N = 5 biological samples. c, m⁶A levels quantified using standard curve in Extended Data Fig. 2d. Bar represents mean. n = 3 or 4 biological replicates. Unpaired two tailed t-test: mammalian bloodstream or insect total RNA vs mRNA P<0.0001; mammalian bloodstream mRNA vs Insect procyclic mRNA P= 0.4162. d, Scatter plot of m⁶A enrichment relative to average transcript expression, expressed as log2 counts per million reads mapped (CPM). Transcripts enriched or depleted in m⁶A IP sample relative to Input sample are indicated in red or blue, respectively. Moderated t-test adjusted with Benjamin Hochberg false discover rate. P values: Supplementary Table 1. Triangles represent VSGs. N = 3 independent IPs. e, Gene set enrichment analysis. Line indicates the enrichment score distribution across VSG genes, ranked according to the log2 fold change between m⁶A-IP and input samples. f, Schematics of oligonucleotides used in RNase H digestion of VSG mRNA and expected digestion products (g). SL: spliced leader; dT: poly deoxi-thymidines. g, m⁶A immunoblotting of mammalian bloodstream forms total RNA digested with RNase H after pre-incubation with indicated oligonucleotides. Methylene Blue stains rRNA. Tub: β-Tubulin. n = 2 independent experiments. h, Mass-spectrometry analysis of total RNA digested independently with enzymes RNase T1 and RNase A. Total RNA was extracted from Trypanosoma brucei (BSF, n = 3; PCF, n = 2), Trypanosoma congolense (n = 2), Trypanosoma cruzi (n = 1), Leishmania infantum (n = 2) and human cells (HEK293T, n = 1). (see also Supplementary Figure S1 and Source Data Figure 1)

Figure 2.. m⁶A is removed from VSG mRNA prior to its degradation.

a, Schematics of VSG mRNA transcript and analyses described in this figure. b, VSG transcript levels (RT-qPCR, pink), m⁶A levels (immunoblotting, light blue) and length of poly(A) tail (PAT assay, dark blue) after transcription halt by actinomycinD (ActD). Data are mean ± s.d. Two-way ANOVA with sidak correction for multiple test. Black asterisks denote significance between mRNA and m⁶A. Grey asterisks denote significance between poly(A) tail and m⁶A (****P<0.0001, *P=0.0104 in mRNA vs m⁶A in 15 min, *P=0.0224 in mRNA vs m⁶A in 30 min, *P=0.0169 in poly(A) vs m⁶A in 30 min). n = 3 transcription inhibition experiments. c, Northern blotting of VSG decay from parasites treated with ActD. Total RNA was incubated with an oligonucleotide located 368 nt upstream of VSG poly(A) tail and digested with RNaseH. Probe hybridizes with conserved 16-mer motif. A0 is the VSG 3’end fragment in which the poly(A) tail was removed by oligo dT-RNase H digestion. Methylene Blue stains rRNA. n = 3 transcription inhibition experiments. Quantification is in Extended Data Figure 4. d, m⁶A immunoblotting of bloodstream form total RNA extracted from parasites treated with ActD (c). Methylene Blue stains rRNA. Quantification is in Extended Data Figure 4. e, VSG transcript levels and m⁶A levels during parasite differentiation from bloodstream to procyclic forms. Total RNA was extracted at different time points after inducing differentiation with cis-aconitate. Same colour code as in Panel b. Data are mean ± s.d. Two-way ANOVA with sidak correction for multiple test (****P<0.0001). n = 3 parasite differentiation experiments. f, m⁶A immunoblotting of parasites differentiating to procyclic forms (e). Methylene blue stains rRNA. (see also Supplementary Figure S1 and Source Data Figure 2)

Figure 3.. Inclusion of m⁶A in the VSG poly(A) tail depends of de novo transcription.

a, Parasites were treated with cis-aconitate (CA), and after washing away compound, parasites were placed in culture in 3 different conditions. Labels 1–5 indicate the conditions at which parasites were collected for immunoblotting analysis (Panel b). Drawings were obtained from smart.servier.com. b, m⁶A immunoblot at each of the 5 conditions (Panel a). n = 3 independent experiments. c, Quantification of immunoblotting in (a). Two-way ANOVA with sidak correction for multiple test. (****P<0.0001. Data are mean ± s.d. Black asterisks refer to condition 3, grey asterisks to condition 5. d, VSG mRNA levels measured by RT-qPCR. Two-way ANOVA with sidak correction for multiple test. (****P<0.0001). Data are mean ± s.d. n = 3 independent experiments. e, m⁶A immunofluorescence analysis. Parasites were treated with Nuclease P1 (NP1) or ActD. Nuclei were stained with Hoechst. Arrows points to weak m⁶A signal. f, Proportion of m⁶A signal in nucleus and cytoplasm. n = 4 experiments with 125 parasites in each. Data are mean ± s.e.m. g, m⁶A levels expressed as mean fluorescence intensity (MFI). Unpaired two tailed t-test (****P<0.0001, *** P= 0.001). Data are mean ± s.d. n = 5 independent experiments, h, RNA-FISH analysis of VSG2 transcripts. Three representative cells are shown. i, Proportion of VSG mRNA signal in nucleus and cytoplasm (h). Data are mean ± s.e.m. n = 5 independent experiments with 34 parasites in each j, m⁶A Dot-Blot of subcellular fractions. Quantity of spotted RNA is indicated. n = 3 fractionation experiments. k, Quantification of dot-blot m⁶A signal (j). Unpaired two tailed t-test P= 0.8753. Data are median. Scale bars, 4μm; DIC, differential interference contrast. (see also Supplementary Figure S1 and Source Data Figure 3)

Figure 4.. Conserved VSG 16-mer motif is required for inclusion of m⁶A in adjacent poly(A) tail.

a, Schematics of VSG double-expressor (DE) cell-lines. VSG117 was inserted in the active bloodstream expression site, which contains VSG2 at the telomeric end. In DE1, VSG117 contains its endogenous 3’UTR with the conserved 16-mer motif (sequence in blue). In DE2, the 16-mer motif of VSG117 was scrambled (sequence in orange). b, Transcript levels of VSG117 and VSG2 transcripts (RT-qPCR), normalized to transcript levels in cell-lines expressing only VSG2. One-way ANOVA with sidak correction for multiple test. (P=0.7612 for VSG2 wt vs VSG117 wt in DE1, ****P<0.0001 for VSG2 wt vs VSG117 mut in DE2). n = 3 independent clones. c, m⁶A immunoblot of mRNA from DE1 and DE2 cell-lines. RNase H digestion of VSG2 mRNA was used to resolve VSG2 and VSG117 transcripts. 50ng and 12.5ng of DE1 were loaded in two separate lanes. n = 3 independent clones. d, m⁶A index calculated as the ratio of m⁶A intensity and mRNA levels, measured in Panels c and b, respectively. und., undetectable. # intensities measured in lane 3 of Panel c. e, m⁶A enrichment in VSG genes. m⁶A-RIP sequencing data was used to calculate, for each VSG gene, the ratio between the number aligned reads in IP versus Input samples. Only VSG transcripts detected in IP sample were used for this analysis. Blue and orange indicate the presence or absence, respectively, of the 16-mer motif in the 3’UTR. Unpaired two sided Mann–Whitney test (P<0,0001,****). f, Scatter plot of m⁶A-RIP enrichment relative to transcript levels of detectable VSG transcripts. Colour code identical to panel e. Dashed lines represents log₂FC=1 and log₂FC=−1. Spearman correlation between the data was R=−0.35. n = 3 for input samples and m⁶A immunoprecipited samples. (see also Supplementary Figure S1 and Source Data Figure 4)

Figure 5.. VSG 16-mer motif inhibits CAF1 and poly(A) tail deadenylation.

a, The length of the VSG poly(A) tail was measured using Poly(A) tailing (PAT) assay after transcription halt by ActD. WT and Mut-16-mer cell-lines were compared. b, VSG117 transcript levels (measured by RT-qPCR, pink) and length of poly(A) tail after transcription halt by ActD. Values were normalized to 0 hour. Two-way ANOVA with sidak correction for multiple test. Black asterisks refer to mRNA, grey asterisks refer to poly(A) tail (****P<0.0001.*** P=0.0002 in VSG117 wt poly(A) tail vs VSG117 mut poly(A) tail in 15 min). Data are mean ± s.d. n = 3 transcription inhibition experiments. c, Length of VSG poly(A) tail upon CAF1 downregulation and after transcription halt by ActD. Poly(A) length was measured by PAT assay. Two-way ANOVA with sidak correction for multiple test. (****P<0.0001). Data are mean ± s.d. n = 3 transcription inhibition experiments. d, VSG transcript levels upon CAF1 downregulation and after transcription halt by ActD. Significance was measured by two-way ANOVA with sidak correction for multiple test. (*P=0.0191). Data are mean ± s.d. n = 3 transcription inhibition experiments. e, RNA-FISH analysis of VSG8 of 4 indicated conditions. DIC, differential interference contrast. Scale represents 4um. f, VSG8 transcript levels expressed as mean fluorescence intensity (MFI) levels of FISH signal. The proportion of nuclear and cytoplasmic staining was calculated as described in Fig. 3. Data are mean ± s.d. Unpaired two-sided t-test (p-value <0,0001,****). n = 3 biological replicates, 100 cells per replicate. (see also Supplementary Figure S1 and Source Data Figure 5)