N6-methyladenosine marks primary microRNAs for processing

Journal name:
Nature
Volume:
519,
Pages:
482–485
Date published:
DOI:
doi:10.1038/nature14281
Received
Accepted
Published online

The first step in the biogenesis of microRNAs is the processing of primary microRNAs (pri-miRNAs) by the microprocessor complex, composed of the RNA-binding protein DGCR8 and the type III RNase DROSHA1, 2, 3, 4. This initial event requires recognition of the junction between the stem and the flanking single-stranded RNA of the pri-miRNA hairpin by DGCR8 followed by recruitment of DROSHA, which cleaves the RNA duplex to yield the pre-miRNA product5. While the mechanisms underlying pri-miRNA processing have been determined, the mechanism by which DGCR8 recognizes and binds pri-miRNAs, as opposed to other secondary structures present in transcripts, is not understood. Here we find in mammalian cells that methyltransferase-like 3 (METTL3) methylates pri-miRNAs, marking them for recognition and processing by DGCR8. Consistent with this, METTL3 depletion reduced the binding of DGCR8 to pri-miRNAs and resulted in the global reduction of mature miRNAs and concomitant accumulation of unprocessed pri-miRNAs. In vitro processing reactions confirmed the sufficiency of the N6-methyladenosine (m6A) mark in promoting pri-miRNA processing. Finally, gain-of-function experiments revealed that METTL3 is sufficient to enhance miRNA maturation in a global and non-cell-type-specific manner. Our findings reveal that the m6A mark acts as a key post-transcriptional modification that promotes the initiation of miRNA biogenesis.

At a glance

Figures

  1. The m6A mark is present in pri-miRNA regions.
    Figure 1: The m6A mark is present in pri-miRNA regions.

    a, Motif discovery analysis in pri-miRNA sequences using FIRE reveals overrepresentation of the METTL3 motif; yellow represents overrepresentation and blue underrepresentation. The magnitude of the over/underrepresentation is reflected in the linear-scale heat map shown at the bottom. The z-score is specified on the right. b, FIRE motif analysis of m6A peaks compared to control sequences of the same length. The overrepresented motif and its z-score is depicted on the right, as in a. c, Density plot of the abundance of m6A marks and their proximity to given miRNAs within transcripts. Peaks obtained from the immunoglobulin G (IgG) immunoprecipitation were used as controls. d, IGV tracks displaying examples of sequencing read clusters from two m6A-seq replicates are shown next to the pre-miRNA genomic loci. The green dots at the bottom of the tracks depict the positions of METTL3 consensus motifs.

  2. METTL3 modulates the expression levels of miRNAs.
    Figure 2: METTL3 modulates the expression levels of miRNAs.

    a, Histogram depicting fold change (log2) in miRNA expression. The ratio of the average value for two independent shRNAs over the average of the two controls is shown. The P value of the analysis is indicated. KD, knockdown. b, Pie-chart representation of the genomic locations of miRNAs downregulated upon METTL3 depletion. c, Heat-map representation of qRT–PCR quantification of eight representative mature miRNAs that were affected by METTL3 depletion. Red represents increased expression while blue represents reduced expression. A heat map depicts their aggregate expression change upon METTL3 modulation. At the bottom, bar graphs showing specific examples. d, Heat-map representation of mature miRNA quantification from c by qRT–PCR upon METTL3 overexpression (OE). e, Heat map representing the quantification of pri-miRNA forms of miRNAs from c and d by qRT–PCR upon METTL3 depletion. All heat maps and bar graphs represent a linear scale. Error bars represent standard deviation (s.d.). P < 5 × 10−4, P < 1 × 10−3.

  3. METTL3 targets pri-miRNAs for m6A methylation.
    Figure 3: METTL3 targets pri-miRNAs for m6A methylation.

    a, FIRE analysis provides motif of METTL3 HITS-CLIP binding sites. The colour scale of the linear scale heat map is the same as in Fig. 1a. b, An example of sequencing clusters obtained from METTL3 HITS-CLIP (blue) and m6A-seq (red). m6A-seq was done in biological duplicate using IgG as control. METTL3 HITS-CLIP was done in biological triplicate. One example is shown for each experiment in the figure. The pink boxes at the bottom of the tracks represent conserved METTL3 motifs. c, Average vertebrate conservation of the METTL3 motif from a group of conserved pri-miRNAs using the PhastCons software17. The dotted green line depicts the average conservation of a region of 100 nucleotides that surrounds (and includes) the motifs. Error bars represent standard error of the mean (s.e.m.). d, Two examples of pri-miRNA genomic regions containing HITS-CLIP tags are shown. At the top, pre-miRNAs are marked in red boxes and METTL3 HITS-CLIP tags in blue boxes. The conserved METTL3 motif is framed in red with the putative methylated adenosine in red.

  4. m6A methylation of pri-miRNAs is required for normal processing by DGCR8.
    Figure 4: m6A methylation of pri-miRNAs is required for normal processing by DGCR8.

    a, In vitro pri-miRNA processing reactions. Pri-let-7e was transcribed with either modified m6A (depicted in the schematic as red dots) or with unmodified bases. b, Hybridization intensities were quantified and normalized to the controls, and are shown in the bar graph. Bars represent the average normalized intensity of three biological replicates. P < 5 × 10−2. c, Input for experiment shown in a. d, Co-immunoprecipitation of the METTL3-interacting protein DGCR8. Western blot using the indicated antibodies with IgG used as control for the immunoprecipitation. e, Immunoprecipitation of endogenous DGCR8. Western blot using an antibody against m6A methylated RNA. f, Similar as e, but control cells or cells depleted of METTL3 were compared. g, Similar immunoprecipitation as e, however, after immunoprecipitation of DGCR8, a radiolabelled RNA linker was ligated to the RNA bound by DGCR8. Bars represent the average normalized intensity of three biological replicates. P < 5 × 10−4. h, Similar as g but the pri-miRNAs bound by DGCR8 were extracted and quantified by qRT–PCR. The bar graph shows the average level of a panel of miRNAs quantified in triplicate and analysed and shown in more detail in Extended Data Fig. 9. P < 1 × 10−4. i, Model of METTL3 regulation of miRNA biogenesis. The molecules represented in the schematic are: histones (yellow), RNA Pol II (orange), METTL3 (green), m6A (red), DGCR8 (blue), DROSHA (pink) and a putative unknown reader of the m6A mark (grey).

  5. METTL3 regulates the expression levels of mature miRNAs.
    Extended Data Fig. 1: METTL3 regulates the expression levels of mature miRNAs.

    a, Unbiased search for cis-regulatory elements using the FIRE algorithm. FIRE motif discovery analysis of pre-miRNAs and pri-miRNA sequences, as well as random sequences of the same length, reveals overrepresentation of the METTL3 motif in pri-miRNAs sequences (containing pre-miRNAs plus adjacent 100 bp) but not in pre-miRNAs. Yellow represents overrepresentation, and blue depicts underrepresentation of the motif. The magnitude of the over/underrepresentation is represented by the linear-scale heat map on the left. A schematic representation of a pre-miRNA and a pri-miRNA is shown on the right. b, c, qRT–PCR (b) and western blot (c) quantifications of METTL3 upon transduction with two independent shRNAs targeting METTL3 (METTL3 KD1 and METTL3 KD2) in MDA-MB-231 cells. Samples were normalized to GAPDH. Data from biological triplicates are shown. Bar graphs represent a linear scale and error bars represent s.d. P < 1 × 10−4, P < 5 × 10−4. d, A volcano plot representation of the microarray of miRNAs shown in Fig. 2a, where the y-axis represents the –log10 of the P value, and the x-axis represents the fold change (log2) between the expression levels of the miRNA from the METTL3 depletion (average of two independent shRNAs) versus the average of two control samples.

  6. Mature miRNAs are downregulated upon METTL3 depletion in MDA-MB-231 cells.
    Extended Data Fig. 2: Mature miRNAs are downregulated upon METTL3 depletion in MDA-MB-231 cells.

    a, Quantification of representative miRNAs that were affected by METTL3 depletion in MDA-MB-231 cells as measured by qRT–PCR. Expression values were normalized to SNORD44 (also known as RNU44). b, An example of a small RNA that did not display expression level changes upon METTL3 knockdown (SNORD44, small nucleolar RNA) normalized to 18S. All experiments were conducted in biological replicates. Bar graphs represent a linear scale and error bars represent s.e.m. P < 5 × 10−4, P < 1 × 10−3, P < 5 × 10−2.

  7. Mature miRNAs are downregulated upon METTL3 depletion in multiple mammalian cell lines.
    Extended Data Fig. 3: Mature miRNAs are downregulated upon METTL3 depletion in multiple mammalian cell lines.

    a, qRT–PCR quantification of examples of miRNAs that were modulated upon METTL3 depletion in HeLa cells. Samples were normalized to RNU44. b, Expression levels of genes used for normalization. All experiments were done in biological replicates. c, d, qRT–PCR (c) and western blot (d) quantifications of METTL3 levels upon transduction with two independent shRNAs targeting METTL3. e, Expression levels of representative miRNAs that were affected by METTL3 depletion in HUVEC cells, as measured by qRT–PCR. Normalization was done by using RNU44 as endogenous control. f, qRT–PCR quantification of METTL3 upon transduction with two independent shRNAs targeting METTL3. g, Quantification of the expression levels of control genes. h, i, Examples of miRNAs affected in mouse embryonic stem cells in which Mettl3 has been targeted using CRISPR26, whose expression levels were measured by qRT–PCR. All experiments were done in biological replicates. Bar graphs represent a linear scale and error bars represent s.d. P < 5 × 10−4, P < 1 × 10−3.

  8. Mature miRNAs are upregulated upon METTL3 over-expression in MDA-MB-231 cells.
    Extended Data Fig. 4: Mature miRNAs are upregulated upon METTL3 over-expression in MDA-MB-231 cells.

    a, qRT–PCR quantification of expression of representative miRNAs modulated upon METTL3 overexpression (METTL3 OE) in MDA-MB-231 cells. Samples were normalized to RNU44. b, qRT–PCR quantification of control RNU44 and GAPDH genes normalized to 18S. All experiments were done in biological replicates. Bar graphs represent a linear scale and error bars represent s.d. P < 5 × 10−4, P < 1 × 10−3.

  9. Quantification of mature and pri-miRNAs levels upon depletion and catalytic inactivation of METTL3 in MDA-MB-231 cells.
    Extended Data Fig. 5: Quantification of mature and pri-miRNAs levels upon depletion and catalytic inactivation of METTL3 in MDA-MB-231 cells.

    a, qRT–PCR quantification of representative pri-miRNAs that were impacted by METTL3 depletion using two independent hairpins in MDA-MB-231 cells. Expression levels were normalized to GAPDH. b, qRT–PCR quantification of GAPDH, endogenous control. All experiments were done in biological replicates. c, d, Quantification of mature (c) and pri-miRNAs (d) upon stable transduction of MDA-MB-231 with either wild-type or a catalytic mutant METTL3. Mature miRNA expression was normalized to RNU44 and pri-miRNAs to GAPDH expression levels. The last bar graph shows the averaged value for all individual miRNAs tested. The experiments were done in biological replicates. Bar graphs represent a linear scale and error bars represent s.d. P < 1 × 10−4, P < 5 × 10−4, P < 1 × 10− 3, P < 5 × 10−2. NS, not significant.

  10. Expression and localization of the Microprocessor upon depletion or overexpression of METTL3.
    Extended Data Fig. 6: Expression and localization of the Microprocessor upon depletion or overexpression of METTL3.

    a, Western blot analysis of METTL3, DROSHA and DGCR8 obtained from nuclear and cytoplasmic fractions of cells transduced with two independent shRNAs targeting METTL3 (shMETTL3 #1 and #2) or with an shRNA control (shC). Tubulin and histone 3 were used as loading controls as well as controls for the efficiency of the fractionation. b, Same as a, but in this case, lysate from cells overexpressing (O/E) METTL3 were compared to wild-type control cells. c, In vitro pri-miRNA processing reactions. Whole-cell extracts of control cells or cells depleted of METTL3 with two independent shRNAs were used to process in vitro transcribed pri-miR-1-1 to produce pre-miR-1-1 in vitro. Pre-miR-1-1 levels were then analysed by northern blot. d, Hybridization intensities of c were quantified, normalized by their inputs and shown in a bar graph format. Bar graphs represent a linear scale and error bars represent s.d.

  11. METTL3 binding and m6A co-localization in pri-miRNA regions.
    Extended Data Fig. 7: METTL3 binding and m6A co-localization in pri-miRNA regions.

    a, FIRE motif discovery analysis of the METTL3 HITS-CLIP binding sites compared to control sequences; two overrepresented versions of the METTL3 motif are shown with a z-score as indicated. The heat map represents a linear scale. b, Venn diagram representation of the overlap of miRNAs affected by METTL3 depletion and bearing the m6A and/or the METTL3 HITS-CLIP tags within 1 kb from any particular miRNA locus. The overlap of miRNAs containing both m6A and METTL3 HITS-CLIP tags is depicted in red. P = 2.4 × 10−15.

  12. m6A facilitates processing of pri-miRNAs.
    Extended Data Fig. 8: m6A facilitates processing of pri-miRNAs.

    a, Schematic representation of the reporters used to study the role of METTL3 in pri-miRNA processing. Represented in red is the pri-let-7e sequence and in green, the control pri-miR-1-1. The top reporter contains a wild-type (WT) sequence of pri-let-7e and the potential sites of methylations are depicted as red dots. The reporter on the bottom contains a mutant version of pri-let-7e in which the five putative adenines of the METTL3 motif were mutated. b, HEK293T cells were transfected with the reporters depicted in a, RNA was extracted, and mature miRNA expression was quantified by qRT–PCR. The bar graph represents the relative expression levels of mature let-7e normalized to mature miR-1-1. c, In vitro binding assays using immunopurified DGCR8. Samples containing in vitro transcribed pri-let-7e with N6-methyladenosine or unmodified adenosines were incubated with magnetic-bead- bound DGCR8, washed, eluted and analysed by northern blot. All reactions contained unmodified pri-miR-1-1 as endogenous control. The top panel shows pri-let-7e and the bottom panel pri-miR-1-1. On the right side of the bar graph is depicted the average intensity of pri-let-7e normalized by pri-miR-1-1 levels. The experiment was done in biological triplicate. Bar graphs represent a linear scale and error bars represent s.d. P < 5 × 10−4. NS, not significant.

  13. METTL3 depletion affects DGCR8 binding to endogenous pri-miRNAs.
    Extended Data Fig. 9: METTL3 depletion affects DGCR8 binding to endogenous pri-miRNAs.

    Immunoprecipitation of endogenous DGCR8 crosslinked to RNA of control cells or cells depleted of METTL3 using two independent shRNAs. After immunoprecipitation, the RNA was extracted and the expression levels of a set of pri-miRNAs were quantified by qRT–PCR. The average quantification is presented in Fig. 4h. Bar graphs represent a linear scale and error bars represent s.d. P < 1 × 10−4, P < 1 × 10−3, P < 1 × 10−2, P < 5 × 10−2.

  14. Schematic depiction of m6A-seq and HITS-CLIP protocols.
    Extended Data Fig. 10: Schematic depiction of m6A-seq and HITS-CLIP protocols.

    a, Schematic representation of the m6A-seq protocol. b, Schematic representation of the HITS-CLIP protocol used. Both protocols are described in detail in Methods.

Accession codes

Primary accessions

Gene Expression Omnibus

References

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Author information

  1. These authors contributed equally to this work.

    • Hyeseung Lee &
    • Hani Goodarzi

Affiliations

  1. Laboratory of Systems Cancer Biology, Rockefeller University, 1230 York Avenue, New York, New York 10065, USA

    • Claudio R. Alarcón,
    • Hyeseung Lee,
    • Hani Goodarzi,
    • Nils Halberg &
    • Sohail F. Tavazoie

Contributions

C.R.A. conceived the project and designed the experiments and S.F.T. supervised the project. C.R.A. performed most of the experiments, H.L. generated stable cell lines, performed qRT–PCR reactions and cloning, H.G. performed computational analyses and N.H. provided technical support. C.R.A. and S.F.T. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

RNA-seq data have been deposited in the Gene Expression Omnibus under accession number GSE60213.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: METTL3 regulates the expression levels of mature miRNAs. (212 KB)

    a, Unbiased search for cis-regulatory elements using the FIRE algorithm. FIRE motif discovery analysis of pre-miRNAs and pri-miRNA sequences, as well as random sequences of the same length, reveals overrepresentation of the METTL3 motif in pri-miRNAs sequences (containing pre-miRNAs plus adjacent 100 bp) but not in pre-miRNAs. Yellow represents overrepresentation, and blue depicts underrepresentation of the motif. The magnitude of the over/underrepresentation is represented by the linear-scale heat map on the left. A schematic representation of a pre-miRNA and a pri-miRNA is shown on the right. b, c, qRT–PCR (b) and western blot (c) quantifications of METTL3 upon transduction with two independent shRNAs targeting METTL3 (METTL3 KD1 and METTL3 KD2) in MDA-MB-231 cells. Samples were normalized to GAPDH. Data from biological triplicates are shown. Bar graphs represent a linear scale and error bars represent s.d. P < 1 × 10−4, P < 5 × 10−4. d, A volcano plot representation of the microarray of miRNAs shown in Fig. 2a, where the y-axis represents the –log10 of the P value, and the x-axis represents the fold change (log2) between the expression levels of the miRNA from the METTL3 depletion (average of two independent shRNAs) versus the average of two control samples.

  2. Extended Data Figure 2: Mature miRNAs are downregulated upon METTL3 depletion in MDA-MB-231 cells. (135 KB)

    a, Quantification of representative miRNAs that were affected by METTL3 depletion in MDA-MB-231 cells as measured by qRT–PCR. Expression values were normalized to SNORD44 (also known as RNU44). b, An example of a small RNA that did not display expression level changes upon METTL3 knockdown (SNORD44, small nucleolar RNA) normalized to 18S. All experiments were conducted in biological replicates. Bar graphs represent a linear scale and error bars represent s.e.m. P < 5 × 10−4, P < 1 × 10−3, P < 5 × 10−2.

  3. Extended Data Figure 3: Mature miRNAs are downregulated upon METTL3 depletion in multiple mammalian cell lines. (371 KB)

    a, qRT–PCR quantification of examples of miRNAs that were modulated upon METTL3 depletion in HeLa cells. Samples were normalized to RNU44. b, Expression levels of genes used for normalization. All experiments were done in biological replicates. c, d, qRT–PCR (c) and western blot (d) quantifications of METTL3 levels upon transduction with two independent shRNAs targeting METTL3. e, Expression levels of representative miRNAs that were affected by METTL3 depletion in HUVEC cells, as measured by qRT–PCR. Normalization was done by using RNU44 as endogenous control. f, qRT–PCR quantification of METTL3 upon transduction with two independent shRNAs targeting METTL3. g, Quantification of the expression levels of control genes. h, i, Examples of miRNAs affected in mouse embryonic stem cells in which Mettl3 has been targeted using CRISPR26, whose expression levels were measured by qRT–PCR. All experiments were done in biological replicates. Bar graphs represent a linear scale and error bars represent s.d. P < 5 × 10−4, P < 1 × 10−3.

  4. Extended Data Figure 4: Mature miRNAs are upregulated upon METTL3 over-expression in MDA-MB-231 cells. (153 KB)

    a, qRT–PCR quantification of expression of representative miRNAs modulated upon METTL3 overexpression (METTL3 OE) in MDA-MB-231 cells. Samples were normalized to RNU44. b, qRT–PCR quantification of control RNU44 and GAPDH genes normalized to 18S. All experiments were done in biological replicates. Bar graphs represent a linear scale and error bars represent s.d. P < 5 × 10−4, P < 1 × 10−3.

  5. Extended Data Figure 5: Quantification of mature and pri-miRNAs levels upon depletion and catalytic inactivation of METTL3 in MDA-MB-231 cells. (264 KB)

    a, qRT–PCR quantification of representative pri-miRNAs that were impacted by METTL3 depletion using two independent hairpins in MDA-MB-231 cells. Expression levels were normalized to GAPDH. b, qRT–PCR quantification of GAPDH, endogenous control. All experiments were done in biological replicates. c, d, Quantification of mature (c) and pri-miRNAs (d) upon stable transduction of MDA-MB-231 with either wild-type or a catalytic mutant METTL3. Mature miRNA expression was normalized to RNU44 and pri-miRNAs to GAPDH expression levels. The last bar graph shows the averaged value for all individual miRNAs tested. The experiments were done in biological replicates. Bar graphs represent a linear scale and error bars represent s.d. P < 1 × 10−4, P < 5 × 10−4, P < 1 × 10− 3, P < 5 × 10−2. NS, not significant.

  6. Extended Data Figure 6: Expression and localization of the Microprocessor upon depletion or overexpression of METTL3. (292 KB)

    a, Western blot analysis of METTL3, DROSHA and DGCR8 obtained from nuclear and cytoplasmic fractions of cells transduced with two independent shRNAs targeting METTL3 (shMETTL3 #1 and #2) or with an shRNA control (shC). Tubulin and histone 3 were used as loading controls as well as controls for the efficiency of the fractionation. b, Same as a, but in this case, lysate from cells overexpressing (O/E) METTL3 were compared to wild-type control cells. c, In vitro pri-miRNA processing reactions. Whole-cell extracts of control cells or cells depleted of METTL3 with two independent shRNAs were used to process in vitro transcribed pri-miR-1-1 to produce pre-miR-1-1 in vitro. Pre-miR-1-1 levels were then analysed by northern blot. d, Hybridization intensities of c were quantified, normalized by their inputs and shown in a bar graph format. Bar graphs represent a linear scale and error bars represent s.d.

  7. Extended Data Figure 7: METTL3 binding and m6A co-localization in pri-miRNA regions. (219 KB)

    a, FIRE motif discovery analysis of the METTL3 HITS-CLIP binding sites compared to control sequences; two overrepresented versions of the METTL3 motif are shown with a z-score as indicated. The heat map represents a linear scale. b, Venn diagram representation of the overlap of miRNAs affected by METTL3 depletion and bearing the m6A and/or the METTL3 HITS-CLIP tags within 1 kb from any particular miRNA locus. The overlap of miRNAs containing both m6A and METTL3 HITS-CLIP tags is depicted in red. P = 2.4 × 10−15.

  8. Extended Data Figure 8: m6A facilitates processing of pri-miRNAs. (167 KB)

    a, Schematic representation of the reporters used to study the role of METTL3 in pri-miRNA processing. Represented in red is the pri-let-7e sequence and in green, the control pri-miR-1-1. The top reporter contains a wild-type (WT) sequence of pri-let-7e and the potential sites of methylations are depicted as red dots. The reporter on the bottom contains a mutant version of pri-let-7e in which the five putative adenines of the METTL3 motif were mutated. b, HEK293T cells were transfected with the reporters depicted in a, RNA was extracted, and mature miRNA expression was quantified by qRT–PCR. The bar graph represents the relative expression levels of mature let-7e normalized to mature miR-1-1. c, In vitro binding assays using immunopurified DGCR8. Samples containing in vitro transcribed pri-let-7e with N6-methyladenosine or unmodified adenosines were incubated with magnetic-bead- bound DGCR8, washed, eluted and analysed by northern blot. All reactions contained unmodified pri-miR-1-1 as endogenous control. The top panel shows pri-let-7e and the bottom panel pri-miR-1-1. On the right side of the bar graph is depicted the average intensity of pri-let-7e normalized by pri-miR-1-1 levels. The experiment was done in biological triplicate. Bar graphs represent a linear scale and error bars represent s.d. P < 5 × 10−4. NS, not significant.

  9. Extended Data Figure 9: METTL3 depletion affects DGCR8 binding to endogenous pri-miRNAs. (117 KB)

    Immunoprecipitation of endogenous DGCR8 crosslinked to RNA of control cells or cells depleted of METTL3 using two independent shRNAs. After immunoprecipitation, the RNA was extracted and the expression levels of a set of pri-miRNAs were quantified by qRT–PCR. The average quantification is presented in Fig. 4h. Bar graphs represent a linear scale and error bars represent s.d. P < 1 × 10−4, P < 1 × 10−3, P < 1 × 10−2, P < 5 × 10−2.

  10. Extended Data Figure 10: Schematic depiction of m6A-seq and HITS-CLIP protocols. (393 KB)

    a, Schematic representation of the m6A-seq protocol. b, Schematic representation of the HITS-CLIP protocol used. Both protocols are described in detail in Methods.

Additional data