Association of the Breast Cancer Protein MLN51 with the Exon Junction Complex via Its Speckle Localizer and RNA Binding Module

Abstract

MLN51 is a nucleocytoplasmic shuttling protein that is overexpressed in breast cancer. The function of MLN51 in mammals remains elusive. Its fly homolog, named barentsz, as well as the proteins mago nashi and tsunagi have been shown to be required for proper oskar mRNA localization to the posterior pole of the oocyte. Magoh and Y14, the human homologs of mago nashi and tsunagi, are core components of the exon junction complex (EJC). The EJC is assembled on spliced mRNAs and plays important roles in post-splicing events including mRNA export, nonsense-mediated mRNA decay, and translation. In the present study, we show that human MLN51 is an RNA-binding protein present in ribonucleoprotein complexes. By co-immunoprecipitation assays, endogenous MLN51 protein is found to be associated with EJC components, including Magoh, Y14, and NFX1/TAP, and subcellular localization studies indicate that MLN51 transiently co-localizes with Magoh in nuclear speckles. Moreover, we demonstrate that MLN51 specifically associates with spliced mRNAs in co-precipitation experiments, both in the nucleus and in the cytoplasm, at the position where the EJC is deposited. Most interesting, we have identified a region within MLN51 sufficient to bind RNA, to interact with Magoh and spliced mRNA, and to address the protein to nuclear speckles. This conserved region of MLN51 was therefore named SELOR for speckle localizer and RNA binding module. Altogether our data demonstrate that MLN51 associates with EJC in the nucleus and remains stably associated with mRNA in the cytoplasm, suggesting that its overexpression might alter mRNA metabolism in cancer. Human metastatic lymph node (MLN)1 51 cDNA was identified from a breast cancer-derived metastatic lymph node cDNA library by differential hybridization of malignant (metastatic lymph node) versus nonmalignant (breast fibroadenoma and normal lymph node) tissues (1). MLN51 presents a correlated pattern of gene amplification and transcript overexpression in breast cancers and cancer-derived cell lines (1-3). In addition, elevated quantities of MLN51 protein have been found in 30% of primary breast tumor samples tested, although no correlation between MLN51 overexpression and a specific histological tumor type or grade has been found (4). MLN51 is a nucleocytoplasmic protein containing, within its amino-terminal half, a coiled-coil domain followed by two nuclear localization signals responsible for its nuclear localization. Its carboxyl-terminal half contains putative Src homology domains 2 and 3 binding sites and mediates its cytoplasmic retention (4). Finally, MLN51 is well conserved during evolution in mammals as well as in more distant species such as worm and fly. From these results, we proposed previously (4) that MLN51 might have a basal cellular function and that its overexpression in cancer cells may have deleterious effects.The MLN51 counterpart in the fly, called Barentsz, has been isolated from a functional genetic screening, as a gene essential for oskar mRNA localization (5). Messenger R...

Splicing remodels messenger ribonucleoprotein architecture via eIF4A3-dependent and -independent recruitment of exon junction complex components

“…Then the prebound MLN51 may join in to form the stable core of the EJC, which in turn can interact with other mRNA-binding proteins that may be already preloaded onto the mRNA, such as Upf3b/3a, to establish a specific mRNP architecture. MLN51 interacts with the EJC component Magoh in a partially RNase-sensitive manner (24). We found that, in the absence of eIF4A3, loading of MLN51 onto mRNA is only slightly reduced, but not abolished, in contrast to loading of Y14/Magoh, which is strongly inhibited.…”

“…eIF4A3 directly contacts the bound RNA in the crystal structure, but Phe-188 of SELOR stacks with an RNA base, suggesting that MLN51 enhances the overall RNA-binding affinity of the complex. Moreover, the SELOR domain by itself can bind RNA independently of splicing (24). GST-SELOR associated with mRNA and pre-mRNA in the presence or absence of eIF4A3 (Fig.…”

EJC core component MLN51 interacts with eIF3 and activates translation

“…1-351), the SELOR domain (MLN51.S, amino acids 137-283) required for MLN51 incorporation into the EJC core (7,22), and the unstructured C-terminal half (MLN51.Ct, amino acids 352-703) implicated in the assembly of stress granules (25). Remarkably, all the truncated proteins carrying the SELOR domain were able to precipitate eIF3 (Fig.…”

“…Given that the SELOR domain is necessary to assemble the EJC core stably onto mRNA (7,22), we wondered whether its interactions with EJC core and eIF3 were mutually exclusive. We tested whether eIF3 still binds MLN51 within the EJC core reconstituted in vitro (22).…”

“…In the presence of ATP, the two RecA domains of eIF4A3 form a large RNA clamp without sequence preference. The most conserved domain of MLN51, named "SELOR" for "SpEckle LOcalizer and RNA-binding module" (7), binds each RecA domain of eIF4A3 and also contacts RNA. Finally, Magoh/Y14 prevents conformational changes of eIF4A3, stabilizing this core complex.…”