7SK methylation by METTL3 promotes transcriptional activity

Abstract

A fundamental feature of cell signaling is the conversion of extracellular signals into adaptive transcriptional responses. The role of RNA modifications in this process is poorly understood. The small nuclear RNA 7SK prevents transcriptional elongation by sequestering the cyclin dependent kinase 9/cyclin T1 (CDK9/CCNT1) positive transcription elongation factor (P-TEFb) complex. We found that epidermal growth factor signaling induces phosphorylation of the enzyme methyltransferase 3 (METTL3), leading to METTL3-mediated methylation of 7SK. 7SK methylation enhanced its binding to heterogeneous nuclear ribonucleoproteins, causing the release of the HEXIM1 P-TEFb complex subunit1 (HEXIM1)/P-TEFb complex and inducing transcriptional elongation. Our findings establish the mechanism underlying 7SK activation and uncover a previously unknown function for the m⁶A modification in converting growth factor signaling events into a regulatory transcriptional response via an RNA methylation-dependent switch.

Fig. 1.. 7SK is m⁶A methylated by METTL3.

(A) Tracks of m⁶A-seq and METTL3 HITS-CLIP on 7SK. The top shows an immunoglobulin G (IgG)–negative control. On the left, a schematic representation of the experiments. Ab, antibody. (B) Isolation of endogenous 7SK by IP of Flag-tagged LARP7. Purified endogenous 7SK as shown by a urea-acrylamide gel (left) and Northern blot (right). IgG was used as controls. MW, molecular weight; nt, nucleotide; NB, Northern blot. (C) Detection of m⁶A in 7SK through UHPLC-MS/MS. The figure shows a representative spectrum from three biological replicates. (D) Knockdown of METTL3 using two independent shRNAs. Total cell extracts were analyzed by Western blot using the indicated antibodies. (E) m⁶A-methylated nuclear RNA was immunoprecipitated and the levels of 7SK were quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Total endogenous 7SK levels were used for normalization. The graph shows means ± SD from three biological replicates. One-way analysis of variance (ANOVA) with Dunnett’s posttest; ****P < 1 × 10⁻⁴. (F) Normalized levels of 7SK were quantified by qRT-PCR. The bar graph depicts the effect of METTL3 depletion using two independent shRNAs. The graph shows means ± SD from three biological replicates. One-way ANOVA with Dunnett’s posttest. (G and H) METTL3 (G) and METTL3/METTL14 protein complexes (H) were expressed in insect cells, affinity-purified using Ni beads, and separated using size exclusion chromatography. The inset shows a representative image of a Coomassie staining of SDS–polyacrylamide gel electrophoresis (SDS-PAGE) from one of the fractions corresponding to the peak (indicated in the chromatogram, absorbance 280 nm was measured in milli-absorbance units (mAU). (I) Methylation of in vitro–transcribed (IVT) 7SK using proteins obtained from (G) and (H). The graph shows means ± SD from a representative experiment out of three biological replicates. One-way ANOVA with Dunnett’s posttest; ****P < 1 × 10⁻⁴.

Fig. 2.. HEXIM1 preferentially binds unmethylated 7SK.

(A) 7SK secondary structure representation. Proteins known to interact with 7SK are depicted as a green (HEXIM1/P-TEFb), gray (HNRNPs), or light blue (LARP7 and MePCE) box. Predicted m⁶A motifs are shown as yellow circles, and the complementary DNA oligo used to fragment 7SK in the RNase H (RH) digestion is shown as a red line. (B) RNase H digestion of endogenous 7SK. Tris-borate EDTA (TBE)–urea acrylamide RNA gel showing untreated 7SK and treated with RNase H, with or without the addition of the complementary DNA oligo (DNA). (C) Relative m⁶A levels in the two fragments generated by RNase H treatment of endogenous 7SK pulled down using Flag-tagged LARP7 as shown in (B). After purification, 7SK was digested by RNase H using the DNA complementary oligo depicted in (A). After fragmentation, the purified fragments were subjected to m⁶A-IP followed by qRT-PCR quantification. The m⁶A-immunoprecipitated 7SK fragments were normalized to each fragment before the m⁶A-IP. The graph shows means ± SD from three biological replicates. Student’s two-tailed t test; ****P < 1 × 10⁻⁴ (D) Stably expressing Flag-tagged proteins were used to pull down endogenous 7SK. After purification of the pulled-down 7SK RNA, a second IP using an m⁶A antibody was performed, and the levels of 7SK were quantified by qRT-PCR. Nonmethylated IVT 7SK RNA was used as a negative control for the m⁶A-IP. The graph shows means ± SD from three biological replicates. One-way ANOVA with Dunnett’s posttest; ***P < 1 × 10⁻³. (E and F) 7SK-protein interaction with HNRNPA2B1 (E) and HEXIM1 (F) upon METTL3 depletion using two independent shRNAs. The top two panels represent the pull-down of endogenous 7SK measured by Northern blot. The immunoprecipitated proteins are detected by Western blot using the indicated antibodies. The bottom three panels show the input protein levels before the IP. Blots are representatives of at least two biological repeats.

Fig. 3.. METTL3 depletion favors HEXIM1 binding to 7SK and decreases EGF-induced transcriptional activation.

(A) m⁶A methylation levels on 7SK upon EGF stimulation in wild-type cells and cells depleted of METTL3. Nuclear m⁶A-IP followed by qRT-PCR quantification of 7SK upon EGF stimulation. A control shRNA and two independent shRNAs targeting METTL3 were stably expressed in HeLa cells. The m⁶A-immunoprecipitated 7SK RNA was normalized to total 7SK. The graph shows means ± SD from three biological replicates. Two-way ANOVA with Tukey’s posttest; ****P < 1 × 10⁻⁴. ns, not significant. (B and C) 7SK-protein interaction with HNRNPA2B1 (B) and HEXIM1 (C) upon EGF stimulation in control cells and cells depleted of METTL3 using two independent shRNAs. The top panels show the pull-down of endogenous 7SK measured by Northern blot. The immunoprecipitated proteins are detected by Western blot using the indicated antibodies. Blots are a representation of two biological replicates. (D) Cellular transcriptional activity upon EGF stimulation in control cells and cells depleted of METTL3 using two independent shRNAs, as measured by RNA Click-iT. Violin plots of fluorescent measurement of 600 cells per condition. (E) Micrographs of representative cell nuclei of (D). Scale bar, 10 μm. Two-way ANOVA with Tukey’s posttest; ****P < 1 × 10⁻⁴. DAPI, 4′,6-diamidino-2-phenylindole; 5-EU, 5-ethynyl uridine.

Fig. 4.. METTL3 phosphorylation prevents its interaction with HEXIM1.

(A) Effect of U0126 on S43-METTL3 phosphorylation induced by EGF in HeLa cells. Western blot analysis of total cell extracts using the indicated antibodies. Blots in (A) to (D) are representatives of three biological repeats. (B) Pull-down of endogenous HEXIM1 from HeLa cell lysates using beads containing GST-tagged unphosphorylated or pS43 METTL3, produced in bacteria. Beads alone were used as a negative control. Western blot analysis using the indicated antibodies. Blots are a representation of three biological repeats. (C) IP of stably expressed Flag-METTL3. The top two panels represent the IP and the bottom three the input. Western blot analysis using the indicated antibodies. Wild-type HeLa cells were used as a negative control (NC) for the IP. (D) IP of stably expressed Flag-HEXIM1.

Fig. 5.. EGF transcriptional activation depends on METTL3 phosphorylation.

(A) Effect of U0126 on the EGF-induced m⁶A methylation of 7SK. The experimental design is similar to Fig. 4A. m⁶A-methylated RNA was immunoprecipitated from cells under the treatments shown in the figure, and 7SK was quantified by qRT-PCR. The m⁶A-immunoprecipitated 7SK RNA was normalized to the total 7SK. The graph shows means ± SD from three biological replicates. Two-way ANOVA with Tukey’s posttest; ****P < 1 × 10⁻⁴. (B) CRISPR-Cas9 mutagenesis of S43A of METTL3. Western blot of METTL3 pS43 upon EGF stimulation in wild-type (WT) cells and two METTL3^S43A homozygous clones. (C) Similar to (A), but in this case, wild-type cells and two METTL3^S43A homozygous clones were used. The graph shows means ± SD from three biological replicates. Two-way ANOVA with Tukey’s posttest; ****P < 1 × 10⁻⁴. (D and E) 7SK-protein interaction with HNRNPA2B1 (D) and HEXIM1 (E), upon EGF stimulation in wild-type cells and two independent METTL3^S43A homozygous clones. Blots are representatives of two biological replicates. (F and G) Effect of U0126 and mutagenesis of S43A-METTL3 on transcriptional activity induced by EGF stimulation, as measured by RNA Click-iT. Violin plots of fluorescent measurement of 600 cells per condition. (G) Micrographs of representative cell nuclei of (F). Scale bar, 10 μm. Two-way ANOVA with Tukey’s posttest; ****P < 1 × 10⁻⁴.

Fig. 6.. 7SK m⁶A methylation is required for HEXIM1 dissociation and transcriptional activation downstream EGF signaling.

(A) CRISPRi was used to down-regulate the expression of 7SK. Once cells were depleted of endogenous 7SK, lentiviral vectors were used to stably express exogenous wild-type or m⁶A-mutant 7SK under the U6 promoter. (B) IP of m⁶A-methylated RNA followed by qRT-PCR of 7SK from cells depleted of 7SK and transduced with wild-type 7SK (red bar) or 7SK m⁶A mutant (blue bar). The m⁶A-immunoprecipitated 7SK RNA was normalized to total 7SK. The graph shows means ± SD from three biological replicates. Student’s two-tailed t test; ****P < 1 × 10⁻⁴ (C) Flag-LARP7 IP of CRISPRi 7SK plus wild-type 7SK or 7SK mutant for m⁶A sites upon EGF stimulation. Western blot for the interacting proteins using the indicated antibodies. Northern blot using a probe against 7SK was performed. (D and E) Effect of mutagenesis of m⁶A sites of 7SK on transcriptional activity induced by EGF stimulation, as measured by RNA Click-iT. Violin plots of fluorescent measurement of 600 cells per condition. (E) Micrographs of representative cell nuclei of (D). Scale bar, 10 μm. Two-way ANOVA with Tukey’s posttest; ****P < 1 × 10^–4. (F) Model. The work presented here is summarized in this model. Upon EGF stimulation, the downstream effector ERK kinase is activated and phosphorylates METTL3 at position S43. This phosphorylation results in the release of METTL3 from its association with HEXIM1, thus allowing the methylation of 7SK. Subsequently, m⁶A methylation of 7SK results in its association with HNRNP proteins and release of the HEXIM1/P-TEFb complex. Free P-TEFb is then available to induce transcription activation.