Phosphorylation of Y14 Modulates Its Interaction with Proteins Involved in mRNA Metabolism and Influences Its Methylation

Abstract

The multicomponent exon junction complex (EJC) is deposited on the spliced mRNA during pre-mRNA splicing and is implicated in several post-splicing events, including mRNA export, nonsensemediated mRNA decay (NMD), and translation control. This report is the first to identify potential post-translational modifications of the EJC core component Y14. We demonstrate that Y14 is phosphorylated at its repeated arginine/serine (RS) dipeptides, likely by SR protein-specific kinases. Phosphorylation of Y14 abolished its interaction with EJC components as well as factors that function downstream of the EJC. A non-phosphorylatable Y14 mutant was equivalent to the wild-type protein with respect to its association with spliced mRNA and its ability in NMD activation, but the mutant sequestered EJC and NMD factors on ribosome-containing mRNA ribonucleoproteins (mRNPs). We therefore hypothesize that phosphorylation of Y14 occurs upon completion of mRNA surveillance, leading to dissociation of Y14 from ribosome-containing mRNPs. Moreover, we found that Y14 is possibly methylated at multiple arginine residues in the carboxyl-terminal domain and that methylation of Y14 was antagonized by phosphorylation of RS dipeptides. This study reveals antagonistic post-translational modifications of Y14 that may be involved in the remodeling of Y14-containing mRNPs.Eukaryotic mRNAs undergo several processing steps before export to the cytoplasm for translation. The splicing reaction removes introns from precursor mRNAs (pre-mRNAs) 4 and positions the exon junction complex (EJC) on spliced mRNA in a sequence-independent manner (1, 2). The EJC is a dynamic multicomponent complex consisting of a heterodimer of Y14 with Mago and a number of associated factors (1, 2). The EJC may be functionally connected to transcription and acts as an adaptor for recruiting factors involved in the RNA metabolism steps downstream of splicing (3).Previous work suggests that the EJC functions for the nuclear export of spliced mRNAs via the interaction of Y14/Mago, as well as other components, with the mRNA export receptor TAP (4 -6). However, depletion of EJC components only marginally affects bulk poly(A) ϩ RNA export in cultured Drosophila cells (7), suggesting that the EJC may be an accessory factor for mRNA export. On the other hand, Y14/ Mago and RNPS1, another EJC component, directly promote NMDmediated mRNA degradation (8, 9). Y14 interacts with the NMD initiator Upf3 in the nucleus, which subsequently recruits other Upf proteins to yield the active NMD complex (1, 2). Depletion of Y14 abolishes NMD, indicating its essential role in this pathway (10). Recent reports show that the EJC promotes efficient translation by enhancing polysome association with mRNAs (11, 12). In particular, Y14/Mago, as well as RNPS1, is implicated in this translation enhancement (11-13). Thus, the EJC participates in several post-splicing events, including mRNA export/surveillance and translation control (1, 2, 14). The Y14/ Mago heterodimer acts as a core that interacts with sever...

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

“…Plasmid Construction-The bacterial expression vectors encoding His-tagged human Y14 and Dcp2, and glutathione S-transferase (GST)-Y14 were described previously (22)(23)(24). The Y14 mutants (W73V, F76V, F93V, Y116V, Y121V, E122V, W148V, and F150V) were generated from pGEX-GST-Y14 by site-directed mutagenesis and were verified by DNA sequencing.…”

“…In Vitro Pull Down Assay-In vitro pull down was performed essentially as described (23,24). PYM was in vitro translated and 35 S-labeled using the TNT-coupled transcription/translation system (Promega) and was incubated with 2 g of individual GST-fusion proteins (ϳ40 pmol) in a 50-l mixture for 30 min at 30°C followed by affinity selection with glutathioneSepharose (GE Healthcare).…”

The RNA Polymerase II C-Terminal Domain Phosphatase-Like Protein FIERY2/CPL1 Interacts with eIF4AIII and Is Essential for Nonsense-Mediated mRNA Decay in Arabidopsis

“…Previous studies have shown that phosphorylation of the Y14 protein, another key component of the EJC, abolished its interaction with other EJC components as well as factors downstream of the EJC (Hsu et al, 2005). Therefore, it is possible that phosphorylation of eIF4AIII similarly disturbs its interaction with other EJC components, causing it to dissociate from the EJC and to diffuse into the cytoplasm, as we observed in the fry2-1 mutant.…”

The Exon Junction Complex Component Y14 Modulates the Activity of the Methylosome in Biogenesis of Spliceosomal Small Nuclear Ribonucleoproteins

“…We have reported previously that the RG-rich sequences in the C-terminal domain of human Y14 can be methylated, and the RS dipeptides can be phosphorylated (6). In this study, our initial attempt to search for the enzymes or regulators responsible for post-translational modification of Y14 led to the identification of the protein-arginine methyltransferase PRMT5 as a potential interacting factor of Y14.…”

“…Y14 contains an RNA recognition motif in the central region, which is involved in the interaction with its stable partner Magoh (5). The C-terminal region of Y14 harboring two consecutive arginine-serine (RS) dipeptides and several arginine and glycine residues is predicted to be less structured but can be post-translationally modified (6). In Drosophila, the Y14/Mago homolog participates in transport and translation control of posterior mRNAs during oogenesis (7,8).…”