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

Dasol Han,Vivian Hoang,Xuemei Zhang,Maxwell Z. Wilson,Kenneth S. Kosik,Andrew P. Longhini,Wendy V. Gilbert

doi:10.1371/journal.pbio.3001535

Abstract

m6A methylation is the most abundant and reversible chemical modification on mRNA with approximately one-fourth of eukaryotic mRNAs harboring at least one m6A-modified base. The recruitment of the mRNA m6A 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 two 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.

Highlights

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
Optogenetic and fluorescent lifetime imaging microscopy (FLIM)-FRET tools, the current study shows that METTL3 undergoes phase separation in nuclei
Based on the previous observation that METTL3/ methyltransferase-like 14 (METTL14) methyltransferase complex can form a dimer of dimer [20], we demonstrated the self-interaction of METTL3 can act as a multivalent scaffold for LLPS (Fig 2D and 2E)

Summary

Introduction

Despite the indispensability of WTAP on m6A modification [21], its interaction dynamics in the writer complex is relatively unexplored compared to METTL3/14 complex 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 interest; SAH, S-adenosylhomocysteine; SAM, Sadenosylmethionine; TCGA, The Cancer Genome Atlas; WTAP, Wilms tumor suppressor-1– associated protein; ZnF, Zinc finger. 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] These findings and studies have been facilitated by optogenetic tools that regulate protein clustering by light stimulation within the live-cell context. With optogenetic and fluorescent lifetime imaging microscopy (FLIM) tools, we demonstrate that cells can utilize LLPS to regulate dynamic assembly of mRNA m6A methyltransferase complex (METTL3/METTL14/WTAP) with stoichiometries that depend on condensate partitioning in a substrate binding–dependent manner

Results

Discussion

Methods