Nuclear export of mRNA is tightly linked to transcription, nuclear mRNA processing, and subsequent maturation in the cytoplasm. Tip-associated protein (TAP) is the major nuclear mRNA export receptor, and it acts coordinately with various factors involved in mRNA expression. We screened for protein factors that associate with TAP and identified several candidates, including RNA helicase DDX3. We demonstrate that DDX3 directly interacts with TAP and that its association with TAP as well as mRNA ribonucleoprotein complexes may occur in the nucleus. Depletion of TAP resulted in nuclear accumulation of DDX3, suggesting that DDX3 is, at least in part, exported along with messenger ribonucleoproteins to the cytoplasm via the TAP-mediated pathway. Moreover, the observation that DDX3 localizes transiently in cytoplasmic stress granules under cell stress conditions suggests a role for DDX3 in translational control. Indeed, DDX3 associates with translation initiation complexes. However, DDX3 is probably not critical for general mRNA translation but may instead promote efficient translation of mRNAs containing a long or structured 5' untranslated region. Given that the DDX3 RNA helicase activity is essential for its involvement in translation, we suggest that DDX3 facilitates translation by resolving secondary structures of the 5'-untranslated region in mRNAs during ribosome scanning.

Coronavirus nucleocapsid protein is abundant in infected cells and participates in viral RNA replication and transcription. The central domain of the nucleocapsid protein contains several arginine/serine (RS) dipeptides, the biological significance of which has not been well investigated. In the present study, we demonstrate that the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated primarily within the RS‐rich region in cells and by SR protein kinase 1 in vitro. The nucleocapsid protein could suppress translation and its RS motif is essential for such an activity. Moreover, phosphorylation of the RS motif could modulate the translation inhibitory activity of the nucleocapsid protein. We further found that RS motif phosphorylation did not significantly affect RNA binding of the nucleocapsid protein but impaired its multimerization ability. We observed that the nucleocapsid protein could translocate to cytoplasmic stress granules in response to cellular stress. Deletion or mutations of the RS motif enhanced stress granule localization of the nucleocapsid protein, whereas overexpression of SR protein kinase 1 inhibited nucleocapsid protein localization to stress granules. The nucleocapsid protein lacking the RS motif formed high‐order RNP complexes, which may also account for its enhanced stress granule localization. Taken together, phosphorylation of the severe acute respiratory syndrome‐CoV nucleocapsid protein modulates its activity in translation control and also interferes with its oligomerization and aggregation in stress granules.

TDP-43 is a highly conserved, 43-kDa RNA-binding protein implicated to play a role in transcription repression, nuclear organization, and alternative splicing. More recently, this factor has been identified as the major disease protein of several neurodegenerative diseases, including frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. For the splicing activity, the factor has been shown to be mainly an exon-skipping promoter. In this study using the survival of motor neuron (SMN) minigenes as the reporters in transfection assay, we show for the first time that TDP-43 could also act as an exoninclusion factor. Furthermore, both RNA-recognition motif domains are required for its ability to enhance the SMN2 exon 7 inclusion. Combined protein-immunoprecipitation and RNA-immunoprecipitation experiments also suggested that this exon inclusion activity might be mediated by multimeric complex(es) consisting of this protein interacting with other splicing factors, including Htra2-␤1. Our data further evidence TDP-43 as a multifunctional RNA-binding protein for a diverse set of cellular activities.TDP-43 is a ubiquitously expressed protein that was originally identified as a factor capable of binding to the TAR DNA of human immunodeficiency virus (1). It was later identified in searches for asymmetrically expressed mouse brain genes 2 and for factor(s) binding to a GU-rich, exon-intron junction sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) 3 pre-mRNA (2). Structurally, TDP-43 contains two RNA-recognition motifs (RRMs), RRM1 and RRM2, and a glycine-rich region in its C terminus. The TDP-43 protein is located primarily in the nucleus (3), and it could act as a transcriptional repressor (1, 3), translational repressor (4), as well as a splicing factor promoting the exon 9 exclusion of the CFTR pre-mRNA (2). As other RRM-containing, RNA-binding proteins (5), the RRM1 of TDP-43 contributes primarily to its RNA-binding ability, whereas RRM2 is needed for correct complex formation (6). The C terminus of TDP-43 including the glycine-rich domain is necessary for formation of heterogeneous nuclear ribonucleoprotein (hnRNP)-rich complexes (7) and for causing the CFTR exon 9 to be skipped during splicing (8). Significantly, TDP-43 was later identified as the pathological signature protein for a number of neurodegenerative diseases, including frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis (ALS), with TDP-43(ϩ), ubiquitin(ϩ) inclusion bodies, or UBI, in the brain/neuron cell (9, 10, and 34). In particular, different miscleaved or modified forms of TDP-43 were trapped in these UBIs (9, 10). The recent analysis of the characteristics of TDP-43 in cultured rodent neurons suggested that the neurodegenerative diseases with TDP-43(ϩ) UBIs likely resulted from the impairment/loss of a spectrum of neuronal functions of TDP-43 because of its trapping within the UBIs (4).The splicing of the transcripts from the...

RNA-binding motif protein 4 (RBM4) plays a regulatory role in alternative splicing of precursor mRNA. We show here that cell stress such as arsenite exposure induces phosphorylation of RBM4 at serine 309 and also drives its cytoplasmic accumulation and targeting to stress granule via the MKK 3/6-p38 signaling pathway. Accordingly, RBM4 suppresses cap-dependent translation in a cis-element-dependent manner. However, RBM4 concomitantly activates internal ribosome entry site (IRES)-mediated translation likely by promoting the association of translation initiation factor eIF4A with IRES-containing mRNAs. Overexpression of RBM4 therefore mimics the effect of cell stress-induced signaling on translation initiation control. Whereas arsenite treatment promotes RBM4 loading onto IRES mRNAs and enhances RBM4 -eIF4A interactions, a nonphosphorylatable mutant of RBM4 was unresponsive to arsenite stress and failed to activate IRES-mediated translation. Thus, our results uncover a previously unrecognized paradigm for the RNA-binding protein RBM4 in its phosphorylation-modulated dual action as a suppressor of cap-dependent and enhancer of IRES-mediated translation in response to stress signals.cell stress ͉ eIF4A ͉ internal ribosome entry site ͉ phosphorylation ͉ splicing factor P osttranscriptional control of eukaryotic gene expression comprises several levels of regulation such as processing, export, turnover, localization, and translation of mRNAs (1). Each regulation step involves various combinations of RNAbinding proteins that form dynamic messenger ribonucleoproteins with the transcript. These messenger ribonucleoproteins may individually play specific roles in mRNA metabolism by forming distinct regulatory complexes (2). Some of them continuously shuttle between the nucleus and cytoplasm and may thus participate in multiple steps of mRNA metabolism in different subcellular compartments. For example, nuclear precursor mRNA splicing factors serine/arginine-rich (SR) proteins were recently implicated in several postsplicing activities including mRNA export, quality control, and translation (1, 3-5).Cellular signaling pathways may relocate messenger ribonucleoproteins and thereby modulate their function. For example, environmental stimuli such as osmotic shock induce phosphorylation and cytoplasmic accumulation of heterogeneous nuclear ribonucleoprotein (hnRNP) A1 via the MAPK pathway and hence alter its activity in splicing regulation (6, 7). Activation of the ERK signaling pathway can drive cytoplasmic accumulation of hnRNP K; blockade of this pathway attenuates the ability of hnRNP K to inhibit translation (8).The cellular response to environmental stress immediately leads to global repression of protein synthesis and aggregation of stalled translation complexes in cytoplasmic foci termed stress granules (SGs) (9, 10). However, stress-induced attenuation of global translation is also accompanied by selective translation of mRNAs that possess internal ribosome entry sites (IRES) (11,12). In particular, IRES-mediated tran...

RBM4 activates exon skipping of PTB transcripts to suppress PTB expression and counteracts PTB-mediated inhibition of alternative splicing during myogenesis.

MicroRNAs (miRNAs) influence many biological processes, including cancer. They do so by posttranscriptionally repressing target mRNAs to which they have sequence complementarity. Although it has been postulated that miRNAs can regulate other miRNAs, this has never been shown experimentally to our knowledge. Here, we demonstrate that miR-107 negatively regulates the tumor suppressor miRNA let-7 via a direct interaction. miR-107 was found to be highly expressed in malignant tissue from patients with advanced breast cancer, and its expression was inversely correlated with let-7 expression in tumors and in cancer cell lines. Ectopic expression of miR-107 in human cancer cell lines led to destabilization of mature let-7, increased expression of let-7 targets, and increased malignant phenotypes. In contrast, depletion of endogenous miR-107 dramatically increased the stability of mature let-7 and led to downregulation of let-7 targets. Accordingly, miR-107 expression increased the tumorigenic and metastatic potential of a human breast cancer cell line in mice via inhibition of let-7 and upregulation of let-7 targets. By mutating individual sites within miR-107 and let-7, we found that miR-107 directly interacts with let-7 and that the internal loop of the let-7/miR-107 duplex is critical for repression of let-7 expression. Altogether, we have identified an oncogenic role for miR-107 and provide evidence of a transregulational interaction among miRNAs in human cancer development.

RNA-binding motif protein 4 (RBM4) has been implicated in the regulation of precursor mRNA splicing. Using differential display analysis, we identified mRNAs that associate with RBM4-containing messenger RNPs in vivo. Among these mRNAs, ␣-tropomyosin (␣-TM) is known to exhibit a muscle cell type-specific splicing pattern. The level of the skeletal muscle-specific ␣-TM mRNA isoform partially correlated with that of RBM4 in human tissues examined and could be modulated by ectopic overexpression or suppression of RBM4. These results indicated that RBM4 directly influences the expression of the skeletal muscle-specific ␣-TM isoform. Using minigenes, we demonstrated that RBM4 can activate the selection of skeletal musclespecific exons, possibly via binding to intronic pyrimidine-rich elements. By contrast, the splicing regulator polypyrimidine tract binding protein (PTB) excluded these exons; moreover, RBM4 antagonized this PTBmediated exon exclusion likely by competing with PTB for binding to a CU-rich element. This study suggests a possible mechanism underlying the regulated alternative splicing of ␣-TM by the antagonistic splicing regulators RBM4 and PTB.

Mutated or dysregulated participates in the progression and metastasis of cancer via its multiple roles in regulating gene expression and cellular signaling. Here, we show that the high expression levels of DDX3 in head and neck squamous cell carcinoma (HNSCC) correlate with lymph node metastasis and poor prognosis and demonstrate that DDX3 is essential for the proliferation, invasion, and metastasis of oral squamous cell carcinoma (OSCC) cells. Microarray analyses revealed that DDX3 is required for the expression of a set of pro-metastatic genes, including ATF4-modulated genes in an aggressive OSCC cell line. DDX3 activated translation of and a set of its downstream targets, all of which contain upstream open reading frames (uORF). DDX3 promoted translation of these targets, likely by skipping the inhibitory uORF. DDX3 specifically enhanced the association of the cap-binding complex (CBC) with uORF-containing mRNAs and facilitated recruitment of the eukaryotic initiation factor 3 (eIF3). CBC and certain eIF3 subunits contributed to the expression of metastatic-related gene expression. Taken together, our results indicate a role for the novel DDX3-CBC-eIF3 translational complex in promoting metastasis. The discovery of DDX3-mediated expression of oncogenic uORF-containing genes expands knowledge on translational control mechanisms and provides potential targets for cancer therapy. http://cancerres.aacrjournals.org/content/canres/78/16/4512/F1.large.jpg .