A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement

Abstract

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers capbinding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis.The post-splicing processing factor Y14 is involved in multiple steps of mRNA biogenesis (1-5). Y14 forms a heterodimer with Mago (6, 7). Y14/Mago genes have coevolved in a wide range of species except for yeast and are essential for germ cell determination during gametogenesis (8). Drosophila Y14/ Mago participates in the transport and translational control of posterior mRNAs during oogenesis (6, 9, 10). In vertebrates, Y14/Mago acts as a core component of the exon junction complex (EJC), 2 which is deposited immediately upstream of every ligated exon during precursor mRNA (pre-mRNA) splicing, and is thus involved in mRNA export, nonsense-mediated mRNA decay (NMD), and translation control (3,5,7,11).Y14 is present in ribosome-associated mRNA ribonucleoprotein (mRNP) fractions (12), and depletion of Y14 inhibits splicing-dependent translational activation (5). In general, the EJC factors act in concert to promote the pioneer round of translation. The Y14/Magoh interacting partner PYM interacts with ribosomal proteins and thus enhances the translation of EJC-bound spliced mRNAs (13). This observation further underscores the importance of Y14 in EJC-mediated translational control. When tethered to a reporter mRNA, Y14 enhances translation, as has been observed with other EJC and NMD factors (11). Y14 may function early during translation, whereas another EJC core factor, eIF4AIII, activates translation after 80S ribosome complex formation (5). Hence, perhaps individual EJC factors modulate productive translation via different mechanisms and in a gene-specific manner.Eukaryotic mRNA decay involves deadenylation-triggered decapping followed by 5Ј to 3Ј exonucleolytic degradation (14,15). Decapping is catalyzed by Dcp2 and is positively and negatively regulated by decapping activators and translation factors, respectively (16 -21). We previously reported that human Y14 interacts with the decap...

Y14 governs p53 expression and modulates DNA damage sensitivity

“…6 ). This slight p53 increase was also observed upon overexpression of FLAG-eIF4A3, but not of the mRNA cap-binding mutant of Y14 (W73V) 4 ( Fig. 4A , lanes 3, 4).…”

“…For example, Y14/Magoh specifically interacts with the methylosome and mRNA decapping complex and thus may regulate the maturation of small nuclear ribonucleoproteins and mRNA stability, respectively 2 3 . In addition, Y14 may selectively promote mRNA translation via binding to the mRNA cap 4 , but its targets remain to be identified.…”

The Interplay between the RNA Decay and Translation Machinery in Eukaryotes

“…Curiously, the Y14 component of the EJC has been suggested to bind the mRNA 5 0 cap as well as the primary decapping enzyme DCP2 through different domains (Chuang et al 2013). Mutating Y14 so that it cannot bind the cap abrogates its ability to support translation, but does not impede the degradation of target mRNAs (Chuang et al 2016). This nicely illustrates the interplay of the EJC with cytoplasmic post-transcriptional processes in determining the propensity of the mRNA to be translated as well as degraded.…”

The exon junction complex regulates the release and phosphorylation of paused RNA polymerase II

“…In contrast to intron definition, this exon definition activity only slightly required the EJC splicing subunits, suggesting an additional mechanism. 21 We now show that the pre-EJC controls exon definition by preventing premature release of Pol splicing via a second mechanism referred to as kinetic coupling. According to the model, decreased elongation rates enhance the recognition of exons containing weak splice sites that would otherwise be skipped by the splicing machinery (Kornblihtt, 2006(Kornblihtt, , 2007.…”