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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.

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Fig. 1: m6A is present in the poly(A) tail of VSG mRNA and other transcripts.
Fig. 2: m6A is removed from VSG mRNA before its degradation.
Fig. 3: Inclusion of m6A in the VSG poly(A) tail depends on de novo transcription.
Fig. 4: Conserved VSG 16-mer motif is required for inclusion of m6A in an adjacent poly(A) tail.
Fig. 5: VSG 16-mer motif inhibits CAF1 and poly(A) tail deadenylation.

Data availability

RNA sequencing datasets of m6A-RIP experiments are deposited in the Sequence Read Archive under the accession code: PRJNA786734. The following panels have associated raw figures: 1b, c, e, h, 2b, c, 3c, d, f, g, i, k, 4b, e, f, 5b–d, f, Extended Data Figs. 2d, e, 3c, 4, 5a–c, 7. The following public databases were used; Trypanosome genome database (TryTrypDB): https://tritrypdb.org/tritrypdb/app; and RNA modifications database (MODOMICS): http://genesilico.pl/modomics/. All data are available on request from the corresponding author. Source data are provided with this paper.

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Acknowledgements

We are grateful to support from the Howard Hughes Medical Institute International Early Career Scientist Program (55007419), a European Molecular Biology Organization Installation grant (2151) and La Caixa Foundation (HR20-00361). This work was also partially supported by the ONEIDA project (LISBOA-01-0145-FEDER-016417) co-funded by Fundos Europeus Estruturais e de Investimento (FEEI) from ‘Programa Operacional Regional Lisboa 2020’ and by national funds from Fundação para a Ciência e a Tecnologia (FCT). S.R.J. was supported by NIH (R35 NS111631). Researchers were funded by individual fellowships from FCT (PD/BD/105838/2014 to I.J.V., 2020.06827.BD to L.S., SFRH/BD/80718/2011 to F.A.-B., PD/BD/138891/2018 to A.T. and CEECIND/03322/2018 to L.M.F.); a Novartis Foundation for Biomedical-Biological research to J.P.d.M.; a Human Frontier Science Programme long-term postdoctoral fellowship to M.D.N. (LT000047/2019); a Marie Skłodowska-Curie Individual Standard European Fellowship to S.S.P. (grant no. 839960); the GlycoPar Marie Curie Initial Training Network (GA 608295) to J.A.R. We thank J. Thomas-Oates (University of York, Centre of Excellence in Mass Spectrometry, Department of Chemistry) for the mass spectrometry analysis. The York Centre of Excellence in Mass Spectrometry was created thanks to a major capital investment through Science City York, supported by Yorkshire Forward with funds from the Northern Way Initiative, and subsequent support from EPSRC (EP/K039660/1 and EP/M028127/1). We also thank A. Temudo, A. Nascimento and A. Lima for bioimaging assistance; the laboratories of A. Tomás and J. Kelly for providing RNA from Leishmania and T. cruzi, respectively; and A. Pena and members of the Figueiredo and Jaffrey laboratories for helpful discussions.

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Contributions

I.J.V., J.P.d.M., L.S., M.D.N., A.T., S.S.P., A.H.M., E.B. and J.A.R. performed the experiments. I.J.V., J.P.d.M., L.S., M.D.N., A.T., S.S.P., A.H.M., E.B., J.A.R., F.A.-B., S.R.J. and L.M.F. planned the experiments and analysed the data. I.J.V., F.A.-B., J.A.R., S.R.J. and L.M.F. conceived the study. I.J.V., S.R.J. and L.M.F. wrote the manuscript, with contributions of all remaining authors.

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Correspondence to Luisa M. Figueiredo.

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Extended data figures and tables

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, a) or insect procyclic stage (PCF, 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 min. 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).

Source data

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 Fig. 1 and Source Data of Extended Figures).

Source data

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

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 Fig. 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).

Source data

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.e.m. 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, 10 µm; 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 Fig. 1 and Source Data of Extended Figures).

Source data

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

a, Overexposure of full immunoblot shown in Fig. 4c. Three independent 16-merWT clones and three independent 16-merMUT 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-merMUT clones. It is also possible to observe a weak VSG2 band in the VSG2 single expressor lane and in the 16-merMUT 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 Fig. 1).

Extended Data Fig. 7 CAF1 depletion.

CAF1 transcript levels measured by RT-qPCR in CAF1 RNAi cell-line used in Fig. 5c–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.

Source data

Extended Data Table 1 Mass-spectrometry features of 34 nucleoside modifications found in T. brucei
Extended Data Table 2 List of oligonucleotides
Extended Data Table 3 Statistical parameters of time course experiments

Supplementary information

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This file contains Supplementary Methods; Supplementary Fig. 1; Supplementary Table 1 legend and Supplementary References.

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Viegas, I.J., de Macedo, J.P., Serra, L. et al. N6-methyladenosine in poly(A) tails stabilize VSG transcripts. Nature 604, 362–370 (2022). https://doi.org/10.1038/s41586-022-04544-0

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