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

Tzu-Wei Chuang,Pey-Jey Peng,Woan-Yuh Tarn

doi:10.1074/jbc.m110.190587

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.

Highlights

Splicing-dependent manner [9, 10]
Cytoplasmic protein ICln (pICln) and MEP50 were detected in the Y14/Magoh pulldown (Fig. 1B), indicating that Y14 may associate with the entire methylosome in the cytoplasm
Y14 May Participate in small nuclear ribonucleoprotein (snRNP) Core Assembly—The above data indicated that Y14/Magoh interacted with the cytoplasmic methylosome and may facilitate Sm protein methylation

Summary

Introduction

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]. 5 ␮g of GST, GST-Y14/ His-Magoh, or any other GST fusion proteins used in this study was incubated with 25 ␮l of HeLa cell nuclear or cytoplasmic extract in a 50-␮l mixture for 30 min at 30 °C followed by affinity selection with glutathione-Sepharose as described [6]. Immunopurified PRMT5 could effectively methylate a recombinant snRNP protein, SmD1, in vitro but not Y14 or Magoh (Fig. 3A, upper panel, lanes 4 – 6).

Results

Conclusion