Abstract
N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic messenger RNA (mRNA) and plays critical roles in RNA biology. The function of this modification is mediated by m6A-selective 'reader' proteins of the YTH family, which incorporate m6A-modified mRNAs into pathways of RNA metabolism. Here, we show that the m6A-binding protein YTHDC1 mediates export of methylated mRNA from the nucleus to the cytoplasm in HeLa cells. Knockdown of YTHDC1 results in an extended residence time for nuclear m6A-containing mRNA, with an accumulation of transcripts in the nucleus and accompanying depletion within the cytoplasm. YTHDC1 interacts with the splicing factor and nuclear export adaptor protein SRSF3, and facilitates RNA binding to both SRSF3 and NXF1. This role for YTHDC1 expands the potential utility of chemical modification of mRNA, and supports an emerging paradigm of m6A as a distinct biochemical entity for selective processing and metabolism of mammalian mRNAs.
Highlights
N6-methyladenosine, the most common internal modification of eukaryotic messenger RNA (mRNA), is associated with the maturation, translation, and eventual decay of protein-coding transcripts (Roundtree et al, 2017; Tuck, 1992)
We further investigated the role of SRSF3 in nuclear export by performing LC-MS/MS on nuclear and cytoplasmic mRNA following knockdown of the protein in HeLa cells
Knockdown of YTHDC1 abolished m6A enrichment by NXF1 as determined by LC-MS/MS (Figure 6C) and reduced transcript enrichment as determined by NXF1 RIP-seq (Figure 6D). These data show that YTHDC1 is required for proper incorporation of methylated mRNA targets into mRNPs consisting of SRSF3 and NXF1, which together function to facilitate nuclear export of their RNA cargo
Summary
N6-methyladenosine, the most common internal modification of eukaryotic mRNA, is associated with the maturation, translation, and eventual decay of protein-coding transcripts (Roundtree et al, 2017; Tuck, 1992). Additional roles in m6A-based regulation of cap-independent translation have been described for eIF3 as well as for the catalytic methyltransferase component METTL3, each functioning to promote the translation of methylated mRNAs (Lin et al, 2016; Meyer et al, 2015) The functions of these ‘reader’ proteins provide a step-wise, mechanistic understanding of the characteristic relationship between m6A modification and mRNA translation and decay. The existence of multifunctional adaptor proteins that have roles in the pre-mRNA splicing and export of mature mRNA suggests that mRNA nuclear processing and transport are regulated by the dynamics of protein–RNA interactions in eukaryotes (Huang and Steitz, 2001; Huang et al, 2003; Muller-McNicoll et al, 2016; Rodrigues et al, 2001; Strasser and Hurt, 2001; Valencia et al, 2008). By performing analysis of subcellular mRNA populations, we show that YTHDC1 utilizes known interactions with SRSF3 to selectively mediate the entry of methylated mRNAs into known routes for export to the cytoplasm
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