The U6 snRNA m6A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention

Abstract

Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N⁶-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis.

Keywords: Intron retention; MAT2A; METTL16; N6-methyladenosine (m(6)A); RNA methylation; S-adenosylmethionine (SAM); SAM metabolism; U6 snRNA; alternative splicing; methionine.

Figure 1. MAT2A Intron Retention Is Regulated

(A) RNA-seq trace of MAT2A from poly(A)-selected total RNA. The retained intron (RI) is highlighted in gray. (B) MAT2A northern blot from Met depletion time course. Data are mean ± standard deviation (SD); n=3. GAPDH serves as loading control. RI and m are MAT2A–RI and mRNA isoforms, respectively. (C) Top, Endogenous MAT2A and GAPDH northern blots with RNA from MAT2A overexpressing cells. Intervening lanes were removed (dashed lines), but the same exposure is shown. Middle, Western blot of MAT2A. The antibody recognizes both endogenous and overexpressed MAT2A. The doublet reflects two posttranslationally modified protein isoforms (α2 and α2´)(Halim et al., 1999). Bottom, quantification of the northern blots; data are mean ± SD; n=3. (D) Northern and western blot analyses after a 12-hr shift to the indicated Met concentrations. Protein is quantified below as mean ± SD; n=3. Unless otherwise noted, all statistical analyses are unpaired Student’s t-tests and significance is annotated as not significant (ns), p≤0.05 (*), p≤0.01 (**), or p≤0.001 (***). Here, comparisons were made to the 100 µM sample. See also Figure S1.

Figure 2. MAT2A Hairpin 1 Is Necessary for Regulation of Intron Retention and Has an N⁶-Methyladenosine Modification

(A) Left, Structure of MAT2A hp1. Red, conserved nonamer; gray circle, predicted m⁶A (A4). Right, Alignment of MAT2A conserved nonamers and U6 snRNA m⁶A site. (B) Diagram of β-globin-MAT2A reporters. Red asterisks, mutation sites. (C) Northern blot and quantification of β-globin reporter assay. Data are mean ± SD; n=4. Statistical analysis compared all –Met samples to the WT-Met control (lane 2). (D) M⁶A-IP experiment with indicated β-globin reporters. RNase H cleavage site is shown as dashed lines; all hp mutants are m9 (see panel B). (E) TLC from an in vitro methylation assay in nuclear extract with uniformly labeled substrates. (F) Same as (E) except the hp1 RNA substrates were radiolabeled at specific adenosines. (G) Same as (F) with A4-labeled WT, C3G, or A9U substrates. See also Figure S2.

Figure 3. METTL16 Methylates the MAT2A Hairpins and Is Required for Splicing Induction

(A) Northern blot of MAT2A after knockdown with control siRNAs (siCtrl), or two METTL16 siRNAs transfected individually or together. Met was depleted for 4 hours. Quantification is mean ± SD; n=4. Statistical analysis compared –Met samples to siCtrl–Met (asterisks), and +Met samples to siCtrl+Met (daggers). (B) Top, Schematic of the MAT2A probes (arrows) and sites of RNase H cleavage (dashed lines). Bottom, Northern blot of m⁶A-IP with poly(A)-selected, RNase H-treated RNA from cells after the indicated siRNA treatments. (C) Formaldehyde RIP of METTL16 with MAT2A RNA. RT-qPCR amplicons are shown; the anti-M16/+Form value for hp1 was set to 1. Data are mean ± SD; n=3. (D) TLC from METTL16 immunodepletion experiment using hp1 radiolabeled at position A4 as substrate. Data are mean ± SD; n=3. (E) In vitro methylation assay using wild-type or mutant rM16-MTD with site-specific radiolabeled hp1 substrates. (F) In vitro methylation assay using wild-type rM16-MTD with A4-labeled WT or mutant substrates. See also Figure S3.

Figure 4. METTL16 Dwell Time on Hairpin 1 Regulates MAT2A Intron Retention

(A) Northern blot of MAT2A after knockdown of METTL16 and overexpression with Flag-tagged siRNA resistant METTL16 proteins (Vec., empty vector; WT, FLAG-METTL16; PP→AA, FLAG-PP185/186AA; F187G, FLAG-F187G). Data are mean ± SD; n=4. Statistical analysis compared all –Met samples to - Met/vector (asterisks), and all +Met samples to +Met/vector (daggers). (B) Same as (A) except β-globin reporters, β-MAT-WT or β-MAT-hp1-C3G, were assayed. Data represented as mean ± SD; n≥3. (C) Diagram of MS2 tethering strategy, representative northern blot, and quantification of intron retention (dark blue, with MS2 binding sites; light blue, no MS2 binding site). The MS2 fusions include a nuclear localization signal (NLS). Data are mean ± SD; n=4. (D) METTL16 native RIP with extracts from cells grown for 3 hr +/−Met. Limited RNA digestion was performed and the MALAT1 amplicon is over 5 kb from the METTL16 binding site, so it serves as a negative control along with GAPDH and β-actin (Brown et al., 2016). Data are mean ± SD; n=3. See also Figure S4.

Figure 5. METTL16 Is the U6 snRNA N⁶-Methyltranfserase

(A) Predicted structures surrounding the MAT2A hp1 and U6 snRNA methylation sites (gray circles). Red, conserved nonamer; Purple, mutants. (B) In vitro methylation with rM16-MTD and indicated hp1 substrates. Data are mean ± SD; n=3. (C) In vitro methylation assay using a site-specifically radiolabeled full-length WT or G88C U6 RNA substrates with rM16-MTD, PP185/186AA, or F187G. (D) Immunodepletion assay (Figure 3D) using a U6 snRNA substrate. Quantification is mean ± SD; n=3. (E) Formaldehyde RIP of METTL16 with U6 and U1 snRNAs. The anti-M16/+Form for U6 was set to 1. Data are mean ± SD; n=3. (F) M⁶A-IP of RNA from two independent colonies of wild-type or ΔDuf890 S. pombe strains. The IP efficiency for the wild-type clone 1 was set to 1. Data are mean ± SD; n=5. See also Figure S5.

Figure 6. Global Analysis of M⁶A After METTL16 Knockdown

(A) Ratio of m⁶A to A in total and poly(A)-selected RNA after METTL16 knockdown; siCtrl was set to 1. Data are mean ± SD; n≥4. (B) Intracellular SAM levels normalized to total ion count (TIC) after METTL16 knockdown. Data are mean ± SD; n=6. (C) Pie chart depicting the annotations of the m⁶A peaks that decrease upon METTL16 knockdown. (D) RNA-seq traces from the m⁶A-seq. The peaks and UACAGAGAA sites are indicated by bars and asterisks, respectively. (E) Formaldehyde RIP as in Figure 3C; the IgG control was set to 1. Data are mean ± SD; n=3. (F) In vitro methylation assay as in Figure 2E. (G) M⁶A-IP was performed on RNA from cells in which MAT2A was knocked down. The m⁶A-IP efficiency was compared to siCtrl samples for a panel of twelve METTL16-dependent and nine METTL16-independent m⁶A peaks (Figure S5E). Each point is the average m⁶A-IP efficiency for a specific peak. (H) Same as (G) except MAT2A was overexpressed. (I) Same as (G) except RNA from siM16-treated cells was assessed +/−MAT2A overexpression. (J) Formaldehyde RIP as in (E). The IP efficiency of the IgG control was set to one, but is not shown. See also Figure S5.

Figure 7. Model For METTL16 Activation Of MAT2A Splicing In Response To SAM Levels

We propose that SAM abundance controls the dwell-time of METTL16 on the MAT2A hp1 by modulating its methylation efficiency. In turn, METTL16 occupancy promotes efficient splicing. See text for details. The diagram depicts posttranscriptional splicing induction, but our data are also consistent with METTL16 promoting co-transcriptional splicing.