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Dynamic assembly of the mRNA m6A methyltransferase complex is regulated by METTL3 phase separation

['Dasol Han', 'Neuroscience Research Institute', 'University Of California', 'Santa Barbara', 'California', 'United States Of America', 'Department Of Molecular', 'Cellular', 'Developmental Biology', 'Andrew P. Longhini']

Date: 2022-02 

m 6 A methylation is the most abundant and reversible chemical modification on mRNA with approximately one-fourth of eukaryotic mRNAs harboring at least one m 6 A-modified base. The recruitment of the mRNA m 6 A methyltransferase writer complex to phase-separated nuclear speckles is likely to be crucial in its regulation; however, control over the activity of the complex remains unclear. Supported by our observation that a core catalytic subunit of the methyltransferase complex, METTL3, is endogenously colocalized within nuclear speckles as well as in noncolocalized puncta, we tracked the components of the complex with a Cry2-METTL3 fusion construct to disentangle key domains and interactions necessary for the phase separation of METTL3. METTL3 is capable of self-interaction and likely provides the multivalency to drive condensation. Condensates in cells necessarily contain myriad components, each with partition coefficients that establish an entropic barrier that can regulate entry into the condensate. In this regard, we found that, in contrast to the constitutive binding of METTL14 to METTL3 in both the diffuse and the dense phase, WTAP only interacts with METTL3 in dense phase and thereby distinguishes METTL3/METTL14 single complexes in the dilute phase from METTL3/METTL14 multicomponent condensates. Finally, control over METTL3/METTL14 condensation is determined by its small molecule cofactor, S-adenosylmethionine (SAM), which regulates conformations of 2 gate loops, and some cancer-associated mutations near gate loops can impair METTL3 condensation. Therefore, the link between SAM binding and the control of writer complex phase state suggests that the regulation of its phase state is a potentially critical facet of its functional regulation.

m 6 A-methylated mRNAs can be recognized by YTH domain containing family proteins (YTHDF1-3) and affected according to the cellular context [ 11 , 12 , 15 , 35 ]. Recent studies have shown that the YTHDFs can undergo LLPS via their IDRs and the phase separation potential can be further facilitated by multiple m 6 A modifications on RNA, suggesting an important role of LLPS on cellular transcriptomic regulation by RNA modification [ 36 – 38 ]. The potential role of LLPS in the mammalian m 6 A writer complex, which has been proposed to interact with nuclear speckles has not been explored. Here, we used immunocytochemistry (ICC) to show that the endogenous writer complex colocalizes with a recognizable phase separated compartment, nuclear speckles. With optogenetic and fluorescent lifetime imaging microscopy (FLIM) tools, we demonstrate that cells can utilize LLPS to regulate dynamic assembly of mRNA m 6 A methyltransferase complex (METTL3/METTL14/WTAP) with stoichiometries that depend on condensate partitioning in a substrate binding–dependent manner.

Membraneless liquid compartments, such as stress granules and processing bodies in cytosol [ 22 ] and nucleoli [ 23 ], Cajal bodies [ 24 ], and nuclear speckles [ 25 ] in nucleus have roles in numerous cellular functions. Often, they are abundant in RNA and affect transcriptomic changes [ 26 , 27 ]. For example, nuclear speckles are important RNA containing membraneless organelles that function as sites for RNA splicing and RNA m 6 A methylation. Liquid–liquid phase separation (LLPS) of these compartments selects biomolecules to become concentrated presumably to implement their functions [ 28 , 29 ]. LLPS is often multiphasic with key molecules selectively partitioning into different layers of individual droplets as seen in nuclear speckles [ 30 ]. LLPS of biomolecules requires multivalent interactions, which are mediated by scaffold proteins with tandem repeat binding sites for other partners [ 31 ] or proteins with intrinsically disordered regions (IDRs, or low-complexity domains), often in concert with nucleic acids [ 31 , 32 ]. Recently, these findings and studies have been facilitated by optogenetic tools that regulate protein clustering by light stimulation within the live-cell context. The use of optogenetic tools, such as photolyase homology region of Arabidopsis thaliana Cry2, lowers the free energy for LLPS of proteins predisposed to phase separate and endows precisely tunable switches for biomolecular interactions that mediate LLPS [ 33 , 34 ]. Key biomolecular interactions are probed rapidly and reliably by artificially linking full-length, as well as mutant and truncated proteins to the Cry2 system and interrogating the system with light-induced clustering.

m 6 A is the most abundant posttranscriptional modification on mRNA [ 1 ], which affects myriad biological processes such as embryonic stem cell differentiation [ 2 – 4 ], brain development [ 5 ], synaptic plasticity [ 6 , 7 ], hematopoiesis [ 8 ], and cancer [ 9 , 10 ]. m 6 A-specific proteins of the YTH domain family (readers) bind modified RNAs to exert m 6 A-dependent regulation of target transcripts, affecting pre-mRNA splicing [ 11 ], translation initiation [ 12 , 13 ], stress granule localization [ 9 ], and RNA export, stability, and decay [ 12 , 14 , 15 ]. mRNA m 6 A modification is mediated by methyltransferase (writer) complex components, methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms tumor suppressor-1–associated protein (WTAP) as characterized by multiple groups elucidating mechanistic insights based on crystal structures and in vitro experiments [ 3 , 16 – 19 ]. METTL3 and METTL14 constitutively interact to form a heterodimer via their methyltransferase domains (MTDs) thereby providing a binding site for RNA, the acceptor substrate [ 3 , 16 – 18 , 20 ]. On the other hand, the donor substrate S-adenosylmethionine (SAM) can only bind METTL3 because the SAM binding pocket of METTL14 has degenerated [ 16 – 18 ]. Despite the indispensability of WTAP on m 6 A modification [ 21 ], its interaction dynamics in the writer complex is relatively unexplored compared to METTL3/14 complex.

Results

Compartmentation of endogenous METTL3 in the nucleus In an effort to explore the role of LLPS in RNA methylation, we explored the association of METTL3, a catalytic subunit of the m6A methyltransferase complex, with nuclear speckles. METTL3 is known to colocalize with nuclear speckle markers as bright punctate densities [20,21,39]. With higher resolution images, METTL3 shows a layered distribution relative the nuclear speckle marker pSC35 (S1A Fig). We characterized the radial distribution of METTL3 around centroids of the nuclear speckle marker pSC35. A clear spike in the METTL3 distribution is seen around the periphery of the pSC35 signal (S1A Fig). This layered organization within the nuclear speckle is consistent with other components of these organelles, specifically spliceosomes, RNA introns, and scaffolding proteins [30]. METTL3 also labeled similar puncta unassociated with nuclear speckles, but with patterns typical of phase separated entities. The multiphasic behavior of METTL3 and its complex compartmentation prompted further experiments into the molecular determinants of METTL3 phase separation.

Nascent RNA is essential for METTL3 opto-condensate formation As METTL3 does not contain a large IDR (S1F Fig) found in some RBPs reported to undergo LLPS [33,41], we sought to investigate the roles of the METTL3 structural and functional domains (Fig 1E) with regard to its LLPS behavior. Recent structural studies acted as a rational design of our mutant METTL3 constructs [16–19]. Deleting the nuclear localization signal (NLS) completely blocked condensate formation, indicating that nuclear environment is required for LLPS of METTL3 (Fig 1F). We rationalized this observation by the fact that m6A writer complex components, such as METTL14, WTAP, and nascent RNA, are largely missing in the cytoplasm. Interaction between METTL3 and METTL14 generates a positively charged groove where mRNAs bind [18]. To test if METTL14 and/or the RNA binding ability of METTL3 is required for the phase separation, we disrupted the METTL14 binding surface of Cry2-mCh-METTL3 by substituting either the whole (residues 462 to 479) or the core residues in the interface loop of METTL3 with alanines (loop to 4A and W475A/N477A). These mutants failed to form condensates indicating that an intact RNA binding groove in the interface loop is necessary for the phase separation of METTL3 (Fig 1G). We next asked if the existence of nascent mRNA molecules is itself important. Because m6A modification on mRNA occurs cotranscriptionally [45], cells were treated with actinomycin D for 4 hours to inhibit transcription and remove nascent RNA in the nucleus. This resulted in a complete absence of METTL3 phase separation (Fig 1H), indicating the necessity of a nascent acceptor substrate RNA. A previous report showed that RNase treatment led to an exclusion of METTL3 from nuclear speckles, further supporting our observations [21]. The leader helix (LH) and 2 type-CCCH Zinc finger (ZnF) domains, which are responsible for WTAP binding and sequence specificity of RNA substrates, respectively [19,46], were dispensable for LLPS revealed by LH deletion- (ΔLH), ZnF disrupting- (C294A for ZnF1, C326A for ZnF2), charge reversal- (R300D/R301D), and aromatic ring-removing (F316A/F321A) mutants (Figs 1I and 1J and S2A). Overall, the nucleus provides a favorable environment to the LLPS of METTL3 by concentrating key components of the methyltransferase complex: METTL14 and nascent RNA (Fig 1K).

Donor substrate-dependent conformations of 2 gate loops in the catalytic site of METTL3 determine LLPS behavior Given that only METTL3, but not METTL14, underwent LLPS, we next focused on their structural difference. In a mRNA m6A writer complex, SAM forms multiple hydrogen bonds with adjacent amino acids in SAM binding pocket of METTL3, whereas the SAM binding pocket in METTL14 is degenerate [16–18]. To test if the donor substrate SAM is required for LLPS, we introduced mutations at SAM binding pocket of Cry2-mCh-METTL3 (D395A, N549A/Q550A). All mutants failed to form condensates while retaining interactions with both METTL3 and METTL14 (Figs 4A and S4A and S4B). These data were highly suggestive that SAM binding could be a crucial factor for phase transition. To exclude the possibility that conformational change caused by the mutation rather than SAM binding affected the LLPS behavior, we removed SAM by treating cells with PF9366, a specific potent inhibitor of SAM synthetase MAT2A (Fig 4F) [50]. Treatment with PF9366 for 4 hours completely prevented Cry2-METTL3 from phase separating (Fig 4B), confirming the necessity of SAM for LLPS. Supplying SAM into PF9366-treated cells rescued the blockage of condensate formation (Fig 4B), ruling out the possibility that the observation was due to a side effect of PF9366. Furthermore, the demixed state of endogenous METTL3 was affected by deprivation or addition of SAM (Fig 4C and 4D). To our knowledge, this is the first demonstration of an endogenous small molecule cofactor exerting control over LLPS behavior of its enzyme. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Ligand binding states determine METTL3 phase transition. (A) Representative images (left) and quantitation (right) representing demixing of Cry2-mCh-METTL3 D395A (catalytic motif, SAM binding pocket) or N549A/Q550A (SAM binding pocket) mutants after (blue dot) blue light stimulation. Scale bar = 10 μm. Gray area (0.09~0.11) is set as threshold of condensate formation. One-sample t test used to compare demixing index above gray area to hypothetical threshold (0.11) ***, P < 0.001, error bars represent mean ± SD (n = 8 for D395A and n = 6 for WT and N549A/Q550A). (B) Representative images (left) and quantitation (right) of effect of SAM depletion from the cells on light-inducible Cry2-mCh-METTL3 condensate formation. PF9366 is pretreated for 4 hours before blue light stimulation. SAM is added 3 hours after PF9366 treatment in presence of PF9366. Scale bar = 10 μm. Gray area (0.09~0.11) is set as threshold of condesate formation. One-sample t test used to compare demixing index above gray area to hypothetical threshold (0.11) ***, P < 0.001, error bars represent mean ± SD (n = 10). (C) Representative images (left) and quantitation (right) representing demixing of endogenous METTL3 at 4 hours after PF9366 treatment. Scale bar = 20 μm. Student t test, ***, P < 0.001, error bars represent mean ± SD (n = 36 for DMSO, n = 34 for PF9366). (D) Representative images (left) and quantitation (right) representing demixing of endogenous METTL3 at 15 minutes after SAM treatment. Scale bar = 20 μm. One-way ANOVA, ***, P < 0.001, error bars represent mean ± SD (n = 15 for 0 mM, n = 18 for 4 mM, and n = 19 for 8 mM). (E) Schematic diagram describing 3 gate loops’ conformations depending on ligand bound states of SAM binding pocket in METTL3. (F) Simplified SAM cycle and chemical inhibitors, PF9366 and ADOX, which block SAM synthesis and SAH hydrolysis, respectively. (G) Effects of small molecule inhibition of SAM cycle on light-inducible Cry2-mCh-METTL3 condensate formation. Scale bar = 10 μm. Gray area (0.09~0.11) is set as threshold of condensate formation. One-sample t test used to compare demixing index above gray area to hypothetical threshold (0.11) ***, P < 0.001, error bars represent mean ± SD (n = 8 for PF9366+ADOX, n = 9 for Untreat, and n = 10 for PF9366). (H) (Top) In vivo EGFP lifetime map of EGFP-METTL3 (wild-type and SAM binding mutants) in HEK293T with light-inducible METTL3 condensates. Scale bar = 10 μm. (Bottom) Color heatmap representing mean fluorescent lifetime values (ns) measured from nucleoplasm without condensates (Nucleoplasm) or inside condensates (Condensates) of each EGFP-fused protein. (I) Histograms showing fluorescent lifetime distribution (>300,000 photons) measured from nucleoplasm without condensates (Nucleoplasm) or inside condensates (Condensates) of each EGFP-fused protein. (J) Representative images (left) and quantitation (right) representing demixing of Cry2-mCh-METTL3 cancer mutants after blue light stimulation. Scale bar = 5 μm. Gray area (0.09~0.11) is set as threshold of condensate formation. One-sample t test used to compare demixing index above gray area to hypothetical threshold (0.11) ***, P < 0.001, error bars represent mean ± SD (n = 9 for WT, n = 7 for R508H, n = 8 for R415C, and n = 10 for E516K). The underlying data for the graphs presented can be found in S1 Values For Plots. METTL3, methyltransferase-like 3; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine. https://doi.org/10.1371/journal.pbio.3001535.g004 Wang and colleagues [17] suggested that the recognition of the adenosine substrate of METTL3 might depend on the ligand binding states of the SAM binding pocket, which, in turn, determines the conformational states of 2 gate loops: gate loop 1 (396 to 411) and gate loop 2 (507 to 515) (Figs 1E and 4E). Gate loop 1 is flipped outwards in the SAM-bound state compared to that in the S-adenosylhomocysteine (SAH)-bound or ligand-free states. Gate loop 2 closes the binding pocket when the SAM or SAH is bound, and undergoes a conformational change in the ligand-free state (Fig 4E) [17]. This led to the hypothesis that the ligand-binding states of SAM binding pocket in METTL3 could differentially affect the phase separation behavior of Cry2-METTL3. Although the SAH hydrolase reaction is reversible and presents a thermodynamic equilibrium that favors SAH synthesis [51], whether the SAM-binding pockets of overexpressed Cry2-METTL3 with PF9366 treatment are SAH-bound or ligand-free states remains unclear. To test if SAH-bound state is capable of LLPS, we pharmaceutically blocked SAH hydrolase activity with adenosine dialdehyde (ADOX) [52] (Fig 4F), causing an accumulation of SAH and the dominant occupation of SAH in the SAM binding pocket together with PF9366 treatment. ADOX/PF9366-treated cells still generated Cry2-METTL3 condensates (Fig 4G), indicating that the SAH-bound state of METTL3 is capable of phase transition. Taken together, among the 3 possible ligand binding states of Cry2-METTL3, SAM-bound (untreated or PF9366+SAM), SAH-bound (PF9366+ADOX), and ligand-free (PF9366), only ligand-free state inhibited condensate formation. Finally, we examined EGFP-fused SAM binding–deficient mutants (D395A and N549A/Q550A) in the presence of Cry2-METTL3 (wild-type) condensates. Interestingly, while retaining the ability to incorporate into condensates, these mutants showed dramatically reduced FRET activities (Fig 4H and 4I), implying that perturbed SAM binding affected the molecular arrangement inside condensates. These data collectively suggest the regulatory role of the ligand binding states on molecular interactions of mRNA m6A writer complex components and, thus, LLPS.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001535

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