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...

The role of Y14 in regulating mRNA decay and P-body formation is described. Y14 interacts with Dcp2 and inhibits mRNA decapping. Moreover, Y14 prevents mRNA degradation and is essential for P-body formation. Y14 may function independent of the EJC to counteract mRNA decay in mRNA metabolism.

SummaryDNA repair deficiency leads to genome instability and hence human disease. Depletion of the RNA processing factor Y14/RBM8A in cultured cells or Rbm8a haplodeficiency in the developing mouse cortex results in the accumulation of DNA damage. Y14 depletion differentially affected the expression of DNA damage response (DDR) factors and induced R-loops, both of which threaten genomic stability. Immunoprecipitation coupled with mass spectrometry revealed DDR factors as potential Y14-interacting partners. Further results confirmed that Y14 interacts with Ku and several DDR factors in an ATM-dependent manner. Y14 co-fractionated with Ku in chromatin-enriched fractions and further accumulated on chromatin upon DNA damage. Y14 knockdown delayed recruitment of DDR factors to DNA damage sites and formation of γH2AX foci and also led to Ku retention on chromatin. Accordingly, Y14 depletion compromised the efficiency of DNA end joining. Therefore Y14 likely plays a direct role in DNA damage repair via its interaction with DDR factors.

The RNA-binding protein Y14 heterodimerizes with Mago as the core of the exon junction complex during precursor mRNA splicing and plays a role in mRNA surveillance in the cytoplasm. Using the Y14/Magoh heterodimer as bait in a screening for its interacting partners, we identified the protein-arginine methyltransferase PRMT5 as a candidate. We show that Y14 and Magoh, but not other factors of the exon junction complex, interact with the cytoplasmic PRMT5-containing methylosome. We further provide evidence that Y14 promoted the activity of PRMT5 in methylation of Sm proteins of the small nuclear ribonucleoprotein core, whereas knockdown of Y14 reduced their methylation level. Moreover, Y14 overexpression induced the formation of a large, active, and small nuclear ribonucleoprotein (snRNP)-associated methylosome complex. However, Y14 may only transiently associate with the snRNP assembly complex in the cytoplasm. Together, our results suggest that Y14 facilitates Sm protein methylation probably by its activity in promoting the formation or stability of the methylosome-containing complex. We hypothesize that Y14 provides a regulatory link between pre-mRNA splicing and snRNP biogenesis.The RNA-binding protein Y14 is evolutionally conserved in metazoans and participates in mRNA biogenesis (1-4). 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). In vertebrates, Y14/Mago, together with another heterodimeric factor eIF4AIII/MLN51, constitutes the core of the exon junction complex (EJC), 2 which is a multiprotein complex assembled on spliced mRNAs in a splicing-dependent manner (9, 10). In the EJC, Y14 directly interacts with several other factors, including RNA export factor (REF/Aly), RNPS1, and Upf3 (11). The EJC serves as a platform for binding of the mRNA export receptor Tip-associated protein (TAP) and factors involved in nonsense-mediated mRNA decay (4, 10, 12). Y14 indeed plays an important role in nonsense-mediated mRNA decay (4).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. PRMT5 is a type II protein methyltransferase that catalyzes both monomethylation and symmetrical dimethylation (13-15). PRMT5 localizes to both the nucleus and the cytoplasm (14, 16). In the nucleus, PRMT5 methylates transcriptional regulatory factors and may thereby affect ...