Specific Y14 domains mediate its nucleo-cytoplasmic shuttling and association with spliced mRNA

Abstract

Pre-mRNA splicing deposits multi-protein complexes, termed exon junction complexes (EJCs), on mRNAs near exon-exon junctions. The core of EJC consists of four proteins, eIF4AIII, MLN51, Y14 and Magoh. Y14 is a nuclear protein that can shuttle between the nucleus and the cytoplasm, and binds specifically to Magoh. Here we delineate a Y14 nuclear localization signal that also confers its nuclear export, which we name YNS. We further identified a 12-amino-acid peptide near Y14's carboxyl terminus that is required for its association with spliced mRNAs, as well as for Magoh binding. Furthermore, the Y14 mutants, which are deficient in binding to Magoh, could still be localized to the nucleus, suggesting the existence of both the nuclear import pathway and function for Y14 unaccompanied by Magoh.

Figure 1. Delineation of the nuclear localization signal (NLS) of Y14.

(A) HeLa cells were transfected with expression vectors encoding myc-tagged pyruvate kinase (PK) fusion proteins. The numbers above each panel correspond to the aa within Y14 that were fused to PK. The subcellular localization of each protein was determined by indirect immunofluorescence using anti-myc monoclonal antibody, 9E10. (B) Schematic drawing of the PK fusion proteins and summary of their intracellular localization as displayed in (A). Also shown is the sequence of the minimally defined Y14 NLS.

Figure 2. Delineation of the nuclear export signal (NES) of Y14.

(A) Expression vectors encoding various nucleoplasmin core (NPc) fusion proteins were transfected into HeLa cells. After expression of the transfected DNAs, the cells were fused with NIH3T3 cells to form heterokaryons and incubated in media containing 100μg/ml cycloheximide for a period of 2 hrs. The cells were then fixed and stained for immunofluorescence microscopy with a monoclonal antibody 9E10 (anti-myc panel) to detect the fusion proteins, and with Hoechst 33258 (Hoechst panel) that discriminates the human and mouse nuclei within the heterokaryon (the mouse nuclei are shown by arrows). The phase panel shows the phase contrast image of the heterokaryons with the cytoplasmic edge highlighted with black dashed lines. (B) Schematic drawing of the NPc fusion proteins and summary of the heterokaryon results as depicted in (A). The sequences of the minimally defined YNS domain are shown below, and the sequences sufficient for NES activity are highlighted in gray shading.

Figure 3. C-terminal of Y14 is also required for binding to Magoh in addition to the RNA-binding domain.

Wild-type and mutant myc-tagged Y14 proteins, produced and labeled with [S]methionine in vitro, were incubated with 5 μg each of GST or GST-Magoh. After washing, bound proteins were eluted, separated on 15% SDS-PAGE gel and visualized by fluorography. The lanes marked Input contain 10% of the total protein used in each binding reaction. The position of molecular mass markers is shown on the left.

Figure 4. Y14 binding to spliced mRNA is Magoh-independent and requires its C-terminus.

(A) Preparation of various recombinant Y14 proteins in E. coli. Wild-type and three mutant proteins were expressed in E. coli cells and purified using Ni-NTA column. Five hundred ng of each purified protein was analyzed by SDS-PAGE. (B) Immunoprecipitation of RNAs from in vitro splicing reaction with Ade2 pre-mRNA in the presence of recombinant proteins was performed. 2.5 μg of anti-T7 tag antibody was used for immunoprecipitation. Pre-mRNAs and mRNA products are indicated with schematic drawings on the right. (C) RNA immunoprecipitation experiment with chicken δ-crystallin pre-mRNA was carried out as in (B).

Figure 5. Subcellular localization of myc-tagged wild-type and mutant Y14 in HeLa cells.

Both wild-type and mutant Y14 are mainly localized in the nucleus. Myc-Y14 cDNA encoding either wild type or several mutants was transfected into HeLa cells, which were subsequently immunostained using anti-myc-tag antibody (9E10) as described in the Materials and Methods.