N-methyladenosine (mA), a ubiquitous RNA modification, is installed by METTL3-METTL14 complex. The structure of the heterodimeric complex between the methyltransferase domains (MTDs) of METTL3 and METTL14 has been previously determined. However, the MTDs alone possess no enzymatic activity. Here we present the solution structure for the zinc finger domain (ZFD) of METTL3, the inclusion of which fulfills the methyltransferase activity of METTL3-METTL14. We show that the ZFD specifically binds to an RNA containing 5'-GGACU-3' consensus sequence, but does not to one without. The ZFD thus serves as the target recognition domain, a structural feature previously shown for DNA methyltransferases, and cooperates with the MTDs of METTL3-METTL14 for catalysis. However, the interaction between the ZFD and the specific RNA is extremely weak, with the binding affinity at several hundred micromolar under physiological conditions. The ZFD contains two CCCH-type zinc fingers connected by an anti-parallel β-sheet. Mutational analysis and NMR titrations have mapped the functional interface to a contiguous surface. As a division of labor, the RNA-binding interface comprises basic residues from zinc finger 1 and hydrophobic residues from β-sheet and zinc finger 2. Further we show that the linker between the ZFD and MTD of METTL3 is flexible but partially folded, which may permit the cooperation between the two domains during catalysis. Together, the structural characterization of METTL3 ZFD paves the way to elucidate the atomic details of the entire process of RNA mA modification.

Ubiquitin (Ub) is an important signaling protein. Recent studies have shown that Ub can be enzymatically phosphorylated at S65, and that the resulting pUb exhibits two conformational states-a relaxed state and a retracted state. However, crystallization efforts have yielded only the structure for the relaxed state, which was found similar to that of unmodified Ub. Here we present the solution structures of pUb in both states obtained through refinement against state-specific NMR restraints. We show that the retracted state differs from the relaxed state by the retraction of the last β-strand and by the extension of the second α-helix. Further, we show that at 7.2, the pK a value for the phosphoryl group in the relaxed state is higher by 1.4 units than that in the retracted state. Consequently, pUb exists in equilibrium between protonated and deprotonated forms and between retracted and relaxed states, with protonated/relaxed species enriched at slightly acidic pH and deprotonated/retracted species enriched at slightly basic pH. The heterogeneity of pUb explains the inability of phosphomimetic mutants to fully mimic pUb. The pHsensitive conformational switch is likely preserved for polyubiquitin, as single-molecule FRET data indicate that pH change leads to quaternary rearrangement of a phosphorylated K63-linked diubiquitin. Because cellular pH varies among compartments and changes upon pathophysiological insults, our finding suggests that pH and Ub phosphorylation confer additional target specificities and enable an additional layer of modulation for Ub signals., a 76-residue signaling protein, is found ubiquitously in cells. Two or more Ub molecules can be covalently linked to form a diubiquitin (diUb) and then a polyubiquitin (polyUb), as an isopeptide bond is formed between the carboxylate group of one Ub (called the distal Ub) and the amine group of another Ub (called the proximal Ub). Owing to the characteristic quaternary structures of polyUb and specific interactions between polyUb and its target proteins (1, 2), a polyUb with a specific linkage can be involved in a distinctive set of cellular functions (3).The heterogeneity of Ub also arises from other types of covalent modifications. Proteomics studies have indicated that Ub is phosphorylated at multiple sites (4, 5). However, PINK1 is the only Ub kinase known to date, which specifically phosphorylates Ub at S65 (6, 7). Under normal conditions, only a fraction of Ub is S65-phosphorylated. However, upon oxidative stress, neurodegeneration, or aging, the level of S65 phosphorylation increases significantly (4, 8). S65-phosphorylated Ub (pUb) in turn can activate PARKIN, a ubiquitin ligase, and induce mitophagy (9-12). However, no other pUb-specific targets have been clearly identified, and how phosphorylation affects Ub signaling in general remains unclear.Previously, Komander and coworkers showed that pUb gives two distinct sets of NMR peaks, which correspond to the two conformational states of pUb exchanging at a slow timescale (13). Based on NMR long-r...

Autophagy receptor p62/SQSTM1 promotes the assembly and removal of ubiquitylated proteins by forming p62 bodies and mediating their encapsulation in autophagosomes. Here we show that under nutrient-deficient conditions, cellular p62 specifically undergoes acetylation, which is required for the formation and subsequent autophagic clearance of p62 bodies. We identify K420 and K435 in the UBA domain as the main acetylation sites, and TIP60 and HDAC6 as the acetyltransferase and deacetylase. Mechanically, acetylation at both K420 and K435 sites enhances p62 binding to ubiquitin by disrupting UBA dimerization, while K435 acetylation also directly increases the UBA-ubiquitin affinity. Furthermore, we show that acetylation of p62 facilitates polyubiquitin chain-induced p62 phase separation. Our results suggest an essential role of p62 acetylation in the selective degradation of ubiquitylated proteins in cells under nutrient stress, by specifically regulating the assembly of p62 bodies.