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

Abstract

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 ...

A Specific Set of Exon Junction Complex Subunits Is Required for the Nuclear Retention of Unspliced RNAs in Caenorhabditis elegans

“…These findings consequently support our results that PRP19 is involved in the nuclear RNA surveillance system. During the methylation of Sm proteins of U snRNPs, association of Y14 with spliceosomal snRNAs was also reported in a mammalian cell line, but in this case, preferential association of U2 snRNA was not observed (83). The EJC subunits may interact with the spliceosome through U2 snRNP at a very early stage in pre-mRNA splicing, because UAP56 was shown to be involved in U2 snRNP recruitment to pre-mRNA (76).…”

The RNA-binding protein Y14 inhibits mRNA decapping and modulates processing body formation

“…The mammalian vectors for expression of the FLAG-tagged proteins Y14, Y14SA, Magoh, MLN51, and eIF4AIII, and the HA-tagged proteins Upf3b and TAP, were described previously (Hsu et al. , 2005; Chuang et al. , 2011).…”

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).…”

“…Purification of Recombinant Proteins-Recombinant GSTfusion and His-tagged proteins were expressed and induced in Escherichia coli strain BLR and purified using glutathione-Sepharose (GE Healthcare) and His⅐Bind Resin (Novagen), respectively, as described (22,23,28).…”