Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase

Abstract

The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.

Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI

“…2b). In addition to the known MTA‐FIP37 interaction (Zhong et al ., 2008), we observed heterodimerization of the Arabidopsis MTA and MTB, consistent with the reported interaction between their mammalian orthologues METTL3 and METTL14 (Liu et al ., 2014; Ping et al ., 2014; Wang et al ., 2014b). We also found that MTB but not MTA formed homodimers in Y2H.…”

“…Orthologues of MTA and MTB interact with each other and with orthologues of FIP37 in S. cerevisiae (Agarwala et al ., 2012) and human cells (Liu et al ., 2014; Ping et al ., 2014; Schwartz et al ., 2014; Wang et al ., 2014b). In S. cerevisiae , writing of m 6 A requires orthologues of MTA (IME4), FIP37 (MUM2) and the yeast‐specific SLZ1 (Agarwala et al ., 2012).…”

“…In mammals, in addition to the orthologues of MTA (METTL3) and FIP37 (WTAP), MTB (METTL14) and the splicing/methylation factor VIR (KIAA1429) are also present in the complex and required for m 6 A writing. Arabidopsis MTA and MTB are nonredundant and, similar to METTL3 and METTL14 (Liu et al ., 2014; Ping et al ., 2014; Wang et al ., 2014b), they also interact directly in Y2H.…”

“…In contrast to S. cerevisiae , where the m 6 A writers reside in nucleoli (Agarwala), their plant orthologues occupy the nucleoplasm, similar to animal systems (Bokar et al ., 1997; Niessen et al ., 2001; Zhong et al ., 2008; Liu et al ., 2014; Ping et al ., 2014). Subnuclear domains, so called nuclear speckles, correspond to sites with active transcription in the interchromatin regions.…”

“…The identity of any of the other proteins that act together with METTL3 remained unknown until it was shown in Arabidopsis that FIP37 (FKBP12 INTERACTING PROTEIN 37) was a partner protein of MTA (the homologue of METTL3) (Zhong et al ., 2008; Shen et al ., 2016). Following this initial discovery, the homologues of FIP37, S. cerevisiae MUM2 (Muddled Meiosis 2), and mammalian WTAP (Wilms tumour 1 associated protein), were shown to interact with METTL3 (MTA) orthologues and to be required for mRNA methylation in their respective model organisms (Agarwala et al ., 2012; Ping et al ., 2014; Wang et al ., 2014b; Liu et al ., 2015). More recently, another human methylase, METTL14, phylogenetically related to METTL3 (Bujnicki et al ., 2002), was shown to form a complex with METTL3 and WTAP, and to be required also for m 6 A formation (Ping et al ., 2014; Wang et al ., 2014b; Liu et al ., 2015).…”