Atomic Mutagenesis of N6-Methyladenosine Reveals Distinct Recognition Modes by Human m6A Reader and Eraser Proteins.

N6-methyladenosine (m6A) is an important modified nucleoside in cellular RNA associated with multiple cellular processes and is implicated in diseases. The enzymes associated with the dynamic installation and removal of m6A are heavily investigated targets for drug research, which requires detailed knowledge of the recognition modes of m6A by proteins. Here, we use atomic mutagenesis of m6A to systematically investigate the mechanisms of the two human m6A demethylase enzymes FTO and ALKBH5 and the binding modes of YTH reader proteins YTHDF2/DC1/DC2. Atomic mutagenesis refers to atom-specific changes that are introduced by chemical synthesis, such as the replacement of nitrogen by carbon atoms. Synthetic RNA oligonucleotides containing site-specifically incorporated 1-deaza-, 3-deaza-, and 7-deaza-m6A nucleosides were prepared by solid-phase synthesis and their RNA binding and demethylation by recombinant proteins were evaluated. We found distinct differences in substrate recognition and transformation and revealed structural preferences for the enzymatic activity. The deaza m6A analogues introduced in this work will be useful probes for other proteins in m6A research.

The m6A reader protein YTHDC2 interacts with the small ribosomal subunit and the 5'-3' exoribonuclease XRN1.

N6-methyladenosine (m6A) modifications in RNAs play important roles in regulating many different aspects of gene expression. While m6As can have direct effects on the structure, maturation, or translation of mRNAs, such modifications can also influence the fate of RNAs via proteins termed "readers" that specifically recognize and bind modified nucleotides. Several YTH domain-containing proteins have been identified as m6A readers that regulate the splicing, translation, or stability of specific mRNAs. In contrast to the other YTH domain-containing proteins, YTHDC2 has several defined domains and here, we have analyzed the contribution of these domains to the RNA and protein interactions of YTHDC2. The YTH domain of YTHDC2 preferentially binds m6A-containing RNAs via a conserved hydrophobic pocket, whereas the ankyrin repeats mediate an RNA-independent interaction with the 5'-3' exoribonuclease XRN1. We show that the YTH and R3H domains contribute to the binding of YTHDC2 to cellular RNAs, and using crosslinking and analysis of cDNA (CRAC), we reveal that YTHDC2 interacts with the small ribosomal subunit in close proximity to the mRNA entry/exit sites. YTHDC2 was recently found to promote a "fast-track" expression program for specific mRNAs, and our data suggest that YTHDC2 accomplishes this by recruitment of the RNA degradation machinery to regulate the stability of m6A-containing mRNAs and by utilizing its distinct RNA-binding domains to bridge interactions between m6A-containing mRNAs and the ribosomes to facilitate their efficient translation.

Human METTL16 is a N6-methyladenosine (m6A) methyltransferase that targets pre-mRNAs and various non-coding RNAs.

N6-methyladenosine (m6A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m6A modifications are installed by the METTL3-METTL14 complex, others appear to be introduced independently, implying that additional human m6A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m6A "writer" protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre-mRNAs. We demonstrate that METTL16 is responsible for N6-methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5' splice sites of pre-mRNAs during splicing, suggesting that METTL16-mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m6A methyltransferase in human cells expands our understanding of the mechanisms by which the m6A landscape is installed on cellular RNAs.

WBSCR22/Merm1 is required for late nuclear pre-ribosomal RNA processing and mediates N7-methylation of G1639 in human 18S rRNA.

Ribosomal (r)RNAs are extensively modified during ribosome synthesis and their modification is required for the fidelity and efficiency of translation. Besides numerous small nucleolar RNA-guided 2'-O methylations and pseudouridinylations, a number of individual RNA methyltransferases are involved in rRNA modification. WBSCR22/Merm1, which is affected in Williams-Beuren syndrome and has been implicated in tumorigenesis and metastasis formation, was recently shown to be involved in ribosome synthesis, but its molecular functions have remained elusive. Here we show that depletion of WBSCR22 leads to nuclear accumulation of 3'-extended 18SE pre-rRNA intermediates resulting in impaired 18S rRNA maturation. We map the 3' ends of the 18SE pre-rRNA intermediates accumulating after depletion of WBSCR22 and in control cells using 3'-RACE and deep sequencing. Furthermore, we demonstrate that WBSCR22 is required for N(7)-methylation of G1639 in human 18S rRNA in vivo. Interestingly, the catalytic activity of WBSCR22 is not required for 18S pre-rRNA processing, suggesting that the key role of WBSCR22 in 40S subunit biogenesis is independent of its function as an RNA methyltransferase.