Journal: Cell

Article Title: Splice site m 6 A methylation prevents binding of U2AF35 to inhibit RNA splicing

doi: 10.1016/j.cell.2021.03.062

Figure Lengend Snippet: Distribution of m 6 A in the worm transcriptome, related to Figure 1 (A) Protein sequence alignment of the methyltransferase domain of METTL16. h, Homo sapiens (NP_076991.3); m, Mus musculus (NP_080473.1); g, Gallus gallus (NP_001026773.1); x, Xenopus laevis (NP_001085334.1); z, Danio rerio (NP_001003611.1); c, Caenorhabditis elegans (NP_499247.2). Secondary structure features from the human METTL16 core methyltransferase domain (PDB: 6GT5 ) are indicated: α helices, β strands and η-3 10 helix. (B) Equimolar amounts of total or poly(A)+ RNA from the adult mouse testes and adult worms ( C. elegans ) were pre-mixed together before performing m 6 A-IP-seq. This allowed us to compare the m 6 A distribution between the species. The worm and the mouse RNAs reveal a similar amount of m 6 A-enriched sequences but only very low number of worm reads pile up as m 6 A peaks. Mean values ± s.d. are plotted (n = 3). (C) Analysis of mouse m 6 A peaks (peak counts are indicated within brackets). (D) Analysis of worm m 6 A peaks (peak counts are indicated within brackets). (E) A consensus sequence identified in the small number (176) of m 6 A peaks identified in worm poly(A)+ RNA. Its significance is not known. (F) RNA-seq analysis of wild-type (WT) and mett-10 ( ok2204 ) knockout (KO) mutant worms showing loss of RNA coverage from the 5′ end of the mett-10 gene in the KO, consistent with the genomic deletion in the mutant. Biological replicas (n = 3) are plotted separately. (G) Multiple worm U6 snRNA transcripts were identified based on sequence homology to mouse Rnu6 . The METTL16/METT-10 methylation consensus sequence and position of m 6 A (red arrowhead) are indicated. (H) Detection of m 6 A methylation in U6 snRNA from total RNA using the SCARLET method . The method allows interrogation of site-specific methylation status (red arrowhead indicates the nucleotide position we examined). The thin-layer-chromatography (TLC) assay used in the protocol is shown. The total RNA is from wild-type (WT) or mett-10 KO worms, grown on nutrient-high or nutrient-low plates. m 6 A, refers to synthetic RNA oligos without (0%) or with (100%) m 6 A , used here as positive controls for the experiment (see ). A part (dotted box) of this image is reproduced as Figure 1 J. (I) The loss of U6 snRNA methylation in the mett-10 KO results in slight increase of cellular U6 snRNA levels. Three input replicas are plotted separately for each tested genotype. (J) The loss of U6 snRNA methylation in the mett-10 KO does not result in overall change in counts of reads covering splice junctions, therefore has no drastic effect on general splicing. Three input replicas are plotted separately for each genotype.

Article Snippet: Polyclonal rabbit anti-METT10D (METTL16) , Abcam , Cat. no. ab186012.

Techniques: Sequencing, RNA Sequencing Assay, Knock-Out, Mutagenesis, Methylation, Thin Layer Chromatography

Journal: Cell

Article Title: Splice site m 6 A methylation prevents binding of U2AF35 to inhibit RNA splicing

doi: 10.1016/j.cell.2021.03.062

Figure Lengend Snippet: Worm METT-10 is an m 6 A writer for U6 snRNA and SAM synthetase mRNA (A) Domain organization of the m 6 A writers: mammalian METTL16 and Caenorhabditis elegans METT-10. MTase, methyltransferase domain; VCR, vertebrate-conserved region. See also Figure S1 A. (B) Quantification of RNA modifications in total and poly(A) + RNA from mouse ( Mus musculus ), insect (silkworm, Bombyx mori ), and worm ( C. elegans ) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The barplot shows the level of m 6 A in poly(A) + RNA. (C) Scheme for mapping m 6 A sites catalyzed by worm METT-10 with m 6 A-IP-seq. Mouse testes RNA is used as an internal control. See also Figure S1 B. (D) The METTL3/METTL14 methylation consensus motif (RRACH) is found on the majority of the mouse m 6 A peaks (total number of peaks in brackets). (E) Meta-analysis of the distribution of m 6 A reads over mouse and worm transcripts. (F) Scheme for identification of m 6 A targets of C. elegans METT-10 by m 6 A-IP-seq. See also Figure S1 F. (G) Based on decreased m 6 A enrichment in mett-10 KO worms compared with the control wild type (WT), we identified the indicated transcripts to be targets of METT-10. See also Figure S1 G. (H) Worm U6 snRNA is enriched in m 6 A-IP with total and poly(A) + RNA, and this enrichment is lost in the mett-10 KO . The normalized counts (reads per million [rpm]) are plotted separately for biological replicates (n = 3). (I) Coverage of m 6 A-enriched reads along the worm U6 snRNA sequence identifies the adenosine (red arrowhead), which is part of the conserved UACm 6 AGAGAA motif, that is methylated. Methylation is lost in mett-10 KO worms. The normalized coverages (rpm) from three biological replicates are plotted separately. (J) Detection of U6 snRNA m 6 A (red arrowhead) in total RNA from WT control or mett-10 KO worms (in biological duplicates). The thin-layer chromatography (TLC) analysis used in the SCARLET method is shown. See also Figure S1 H.

Article Snippet: Polyclonal rabbit anti-METT10D (METTL16) , Abcam , Cat. no. ab186012.

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Methylation, Sequencing, Thin Layer Chromatography

Journal: Cell

Article Title: Splice site m 6 A methylation prevents binding of U2AF35 to inhibit RNA splicing

doi: 10.1016/j.cell.2021.03.062

Figure Lengend Snippet: A conserved stem-loop structure containing the 3′ splice site identifies it for methylation by METT-10 (A) Position of m 6 A marks introduced by human METTL16 on the 3ʹ UTR of human MAT2A SAM synthetase as well as C. elegans METT-10 on the 3ʹ splice site of worm sams-3 pre-mRNA. A 30-nt RNA fragment (RNA-1; ) spanning the intron 2-exon 3 boundary of the worm sams-3 gene is predicted to fold into a stem-loop structure, with the METT-10 methylation motif UACm 6 AGAAAC (red) present in the loop region. This is very similar to the substrate requirement of mammalian METTL16. (B) Purification of recombinant worm METT-10 and human METTL16 proteins for in vitro methylation assays. Shown are in vitro methylation assays with METT-10 and the indicated RNA substrates, based on the sams-3 intron 2/exon 3 junction sequence, using radioactive 14 C-SAM as a methyl donor. The UACAGAAAC motif (red) and residues that were mutated (blue) are highlighted. The reaction products were resolved by PAGE and exposed to detect the radioactivity ( 14 C) signal. (C) In vitro methylation with recombinant METT-10 and the RNA substrates, based on the sams-3 intron 2/exon 3 junction sequence, carrying mutations in the stem region. (D) Splicing of WT and mutant (MUT) transgene reporter constructs injected into worm gonads. A MUT construct with triple mutations (AAC→CUU) within the methylation consensus motif (in the exon 3 part) increases 3′ splice site use, producing higher amounts of the PC isoform. Barplots depict the mean relative proportion of individual isoforms ± SD (n = 4). The p values were calculated using t tests. ∗ p ≤ 0.05, ∗∗∗ p ≤ 0.001. See also Figure S3 A for transgene analysis in the mett-10 KO background. (E) METT-10 consensus motif (red) and regions allowing secondary structure formation (yellow) are conserved in various worm species. Changes (green) in C. japonica are compensatory. (F) Sequence alignment of the genomic region at the intron-exon boundary of the SAM synthetase gene from different organisms. The METT-10/METTL16 methylation consensus motif is highlighted (blue). Shown are in vitro methylations with ~30-nt RNAs corresponding to the intron-exon boundary sequence, carried out with recombinant human METTL16. The reaction products were resolved by PAGE and exposed to detect the radioactivity ( 14 C) signal. See also Figure S3 B for the same reactions carried out with worm METT-10.

Article Snippet: Polyclonal rabbit anti-METT10D (METTL16) , Abcam , Cat. no. ab186012.

Techniques: Methylation, Purification, Recombinant, In Vitro, Sequencing, Radioactivity, Mutagenesis, Construct, Injection

Journal: Cell

Article Title: Splice site m 6 A methylation prevents binding of U2AF35 to inhibit RNA splicing

doi: 10.1016/j.cell.2021.03.062

Figure Lengend Snippet: m 6 A methylation of a specific 3′ ss in SAM synthetase pre-mRNA requires a stem-loop structure, related to Figure 3 (A) Wild-type (WT) transgene reporter constructs based on the sams-3 gene were injected into mett-10 KO worm gonads and multiple independent progeny lines stably expressing them were derived. Splicing patterns were analyzed by RT-PCR analysis using primers specific to the reporter. A mutated (MUT) construct with triple mutations (AAC →CUU) within the methylation consensus motif (in the exon3) was also tested. Lack of 3′ splice site m 6 A methylation in the KO worms results in similar isoform levels from both WT and MUT constructs. Barplots depict mean relative proportion of individual isoforms ± s.d. (n = 3). See also Figure 3 D. PC, protein-coding; AS, alternatively spliced; IR, intron-retained. (B) In vitro methylation assay using recombinant worm METT-10 protein and synthetic RNAs. The RNAs correspond to the intron-exon boundary of the SAM synthetase pre-mRNA from the indicated organisms, where the 3′ splice has the METT16/METT-10 methylation consensus motif. Note that the corresponding intron-exon boundary sequence in mouse Mat2a pre-mRNA has no consensus motif, unlike the confirmed METTL16 target site in its 3′ UTR. See also Figure 3 F for the in vitro methylations with human METTL16. It appears that the worm METT-10 is inefficient on targets other than its own sams target site, while human METTL16 is active on all targets carrying the methylation consensus motif.

Article Snippet: Polyclonal rabbit anti-METT10D (METTL16) , Abcam , Cat. no. ab186012.

Techniques: Methylation, Construct, Injection, Stable Transfection, Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, In Vitro, Recombinant, Sequencing

Journal: Cell

Article Title: Splice site m 6 A methylation prevents binding of U2AF35 to inhibit RNA splicing

doi: 10.1016/j.cell.2021.03.062

Figure Lengend Snippet: The 3′ ss m 6 A methylation-mediated splicing inhibition is conserved in human cells, related to Figure 5 (A) In vitro methylation assay using recombinant human METTL16 or worm METT-10 proteins and radioactive 14 C-SAM as the methyl donor, using RNAs (two different lengths) corresponding to the intron 2-exon 3 boundary of the worm sams-3 gene. The methylation consensus motif (red) and target adenosine (in bold) are shown. The reaction products were resolved by PAGE, the gel was stained with Methylene Blue to reveal the RNAs (to assure similar levels), and exposed to detect the radioactivity signal ( 14 C). The human METTL16 is able to recognize and methylate the worm sams-3 target site, allowing us to test worm transgene reporter constructs in human HeLa cells. See also Figure 5 A, and below. (B) RT-PCR analysis of the transcripts produced from worm sams-3 transgene construct transfected into HeLa cells. Wild-type (WT) construct with the 3′ splice site which can be methylated by human METTL16 shows different splicing pattern when compared to the construct with mutations (MUT: AAC→CUU) in the methylation consensus motif (on the part that sits on exon 3). Compare ratios of alternatively spliced (AS) and correctly spliced protein-coding (PC) isoforms. Three biological replicates, each with three technical replicates, were used to quantify the individual isoforms and produce the barplot in Figure 5 A. Part of this panel (replicate #1, dotted box) is reproduced in Figure 5 A. (C) In vitro splicing assay shows that an artificially introduced 3′ splice site (3′ ss) m 6 A within the human beta-globin pre-mRNA abolishes its splicing in human HeLa nuclear extracts, with neither the fully spliced product nor the lariat intermediate being detected. Presence of a single exonic m 6 A has no effect on splicing. See also Figure 5 B. A major RNA band (indicated with an asterisk) below the unspliced RNA substrate is an irrelevant non-ligated species leftover from production of the splint-ligated RNA substrate (see )

Article Snippet: Polyclonal rabbit anti-METT10D (METTL16) , Abcam , Cat. no. ab186012.

Techniques: Methylation, Inhibition, In Vitro, Recombinant, Staining, Radioactivity, Construct, Reverse Transcription Polymerase Chain Reaction, Produced, Transfection, Splicing Assay

Journal: Cell

Article Title: Splice site m 6 A methylation prevents binding of U2AF35 to inhibit RNA splicing

doi: 10.1016/j.cell.2021.03.062

Figure Lengend Snippet: Creation of a mouse KI and cKO mutants for Mettl16 , related to Figure 6 (A) Strategy for generation of the Mettl16 knockin (KI) point mutant mice. A part of the genomic sequence of the Mettl16 exon 5 and the predicted protein sequence encoded are shown. A single guide RNA (gRNA) targeting this region was used to guide Cas9 endonuclease activity and homology-mediated repair to introduce nucleic acid mutations that eventually result in the following amino acid changes: F187G (RNA binding mutant) and PP185-186AA (catalytic dead mutant). Sequence of part of the repair templates bringing the mutations (in red) are shown. (B) Examples of Sanger sequencing of genomic PCR to detect the WT, F187G and PP185-186AA Mettl16 alleles (from mouse tail DNA). Representative ethidium bromide-stained agarose gels showing resolved PCR products is shown. Primer sequences are provided in . (C) Strategy for creation of the floxed Mettl16 allele. The mouse line with floxed Mettl16 allele and an inserted FRT-flanked selection markers cassette (LacZ and neomycin) was obtained from the KOMP repository at UC, Davis. Animals were crossed to remove the selection markers (STAR Methods). Using further crosses, we then brought together the floxed ( Mettl16 loxP ) allele and the Mettl16 null allele ( Mettl16 - ). Crosses between Mettl16 loxP/- and Mettl16 loxP/+ ; vasa-Cre partners gave us the Mettl16 loxP/- ; vasa-Cre mice = conditional knockout (cKO) mutant. In the cKO, the gene is deleted in the male and female germline (starting from embryonic day E14.5 in the male germline). Representative ethidium bromide-stained agarose gels showing resolved PCR products detecting the different alleles and Cre driver is shown. Primer sequences are provided in .

Article Snippet: Polyclonal rabbit anti-METT10D (METTL16) , Abcam , Cat. no. ab186012.

Techniques: Knock-In, Mutagenesis, Sequencing, Activity Assay, Introduce, RNA Binding Assay, Staining, Selection, Knock-Out

Journal: Cell

Article Title: Splice site m 6 A methylation prevents binding of U2AF35 to inhibit RNA splicing

doi: 10.1016/j.cell.2021.03.062

Figure Lengend Snippet: RNA m 6 A methylation activity of mouse METTL16 is essential for development and has the potential to methylate the 3′ ss of target RNAs (A) Analysis of knockin (KI) mouse mutants for Mettl16 , with mutations abolishing catalytic activity or RNA binding. A structural model of human METTL16 (PDB: 6GFK ) shows the two prolines (PP185–PP186) of the NPPF catalytic motif close to the bound SAH molecule, and a model of human METTL16 in complex with bound MAT2A hairpin RNA (PDB: 6DU4 ) shows the F187 that flips in to interact with the target adenosine upon substrate RNA binding. Introduced mutations are indicated. See also A and S7B. Shown are genotypes of animals recovered in born litters from crosses between heterozygous Mettl16 knockin (KI) parents ( Mettl16 KI/+ ). Homozygous KI mutants were not obtained for either mutation, indicating lethality. HET, heterozygous; HOM, homozygous KI. (B) Multiple-tissue western blot showing tissue-specific expression of mouse METTL16. A loading control is provided by detection of PARK7. (C) Representative picture of atrophied testes from a mouse with conditional ( Vasa -Cre) deletion of Mettl16 in the germline. Such animals are infertile. See also Figure S7 C. (D) Histology of adult mouse testes showing complete absence of germ cells in seminiferous tubules from mice with conditional ( Vasa -Cre) deletion of Mettl16 in the germline. cKO, conditional KO. The control HET testis shows all different stages of germ cells, including post-meiotic round spermatids and elongated spermatids. (E) Scheme showing identification of putative targets of mammalian METTL16 on 3′ ss. The total numbers of 3′ ss checked and those recovered with the METTL16/METT-10 motifs are given. The predicted secondary structure of one such RNA (intron-exon boundary with 3′ ss) is shown. (F) In vitro methylation assays with recombinant human METTL16 and the indicated RNAs. The RNA sequence for mouse Mat2a is from the 3′ UTR, whereas for other mouse genes it spans the intron-exon boundary (sequences are shown below). Reactions were resolved by PAGE, and the radioactivity ( 14 C) signal was detected. (G) Two transcripts that show increased splice junction reads specifically in Mettl16 KO embryos (morulae at E2.5 or blastocysts at E3.5), indicating increased use of that ss in the absence of METTL16. Genomic coordinates of the 3′ ss and the underlying sequence on the Crick strand are shown. In vitro methylation assays with RNAs spanning the intron-exon boundary show methylation of the 3′ ss by mammalian METTL16.

Article Snippet: Polyclonal rabbit anti-METT10D (METTL16) , Abcam , Cat. no. ab186012.

Techniques: Methylation, Activity Assay, Knock-In, RNA Binding Assay, Mutagenesis, Western Blot, Expressing, In Vitro, Recombinant, Sequencing, Radioactivity

Journal: Cell

Article Title: Splice site m 6 A methylation prevents binding of U2AF35 to inhibit RNA splicing

doi: 10.1016/j.cell.2021.03.062

Figure Lengend Snippet: Conserved targets of METTL16-mediated m 6 A methylation activity and specialization of the C-terminal VCR in vertebrates SAM levels are highly regulated in vivo , and this is achieved by splicing regulation of the SAM synthetase RNA ( sams-3 or MAT2A ). Under high-SAM conditions, METT-10 m 6 A methylates a 3ʹ ss in sams-3 pre-mRNA to directly inhibit splicing, whereas methylation in the 3ʹ UTR of MAT2A by mammalian METTL16 leads to intron retention/decay of the RNA. Under low-SAM conditions, mammalian METTL16 binds hairpins in the 3ʹ UTR of MAT2A and uses its C-terminal VCR to stimulate splicing of the terminal intron, whereas in nematodes, absence of ss methylation allows normal splicing to proceed. The different mechanisms also highlight the different approaches to regulation of SAM levels: nematode METT-10 turns off SAM production, whereas mammalian METTL16 actively turns on SAM production.

Article Snippet: Polyclonal rabbit anti-METT10D (METTL16) , Abcam , Cat. no. ab186012.

Techniques: Methylation, Activity Assay, In Vivo

Journal: Cell

Article Title: Splice site m 6 A methylation prevents binding of U2AF35 to inhibit RNA splicing

doi: 10.1016/j.cell.2021.03.062

Figure Lengend Snippet:

Article Snippet: Polyclonal rabbit anti-METT10D (METTL16) , Abcam , Cat. no. ab186012.

Techniques: Recombinant, Diagnostic Assay, Western Blot, Protease Inhibitor, Multiplex Assay, Gel Extraction, Purification, DC Protein Assay, Sequencing, Expressing, Knock-Out, Mutagenesis, Generated, Software, Microscopy