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...

shRNA loss-of-function screens were used to identify kinases that were rate-limiting for promoting cell proliferation and survival. Here, we study the differences in kinase requirements among various human cells, including freshly prepared primary cells, isogenic cells, immortalized cells, and cancer cell lines. Closely related patterns of kinase requirements among the various cell types were observed in three cases: (i) in repeat experiments using the same cells, (ii) with multiple populations of freshly prepared primary epithelial cells isolated from the same tissue source, and (iii) between nearly isogenic cells that differ from each other by the expression of a single gene. Other commonly used cancer cell lines were distinct from one another, even when they were isolated from similar tumor types. Even primary cells of different lineages isolated from the same tissue source showed many differences. The differences in kinase requirements among cell lines observed in this study suggest that the control of proliferation and survival may be significantly different between cell lines and that simple comparisons from any one cell to another may be misleading. Although the regulation of cell proliferation and survival are heavily studied areas, we did not see a bias in these screens toward the identification of previously known and well studied kinases, suggesting that our knowledge of molecular events in these areas is still meager.cancer ͉ essential kinases ͉ shRNA screens ͉ fingerprints T he introduction of siRNAs into cells either by transfection of siRNAs themselves or processing shRNAs into their active siRNA form within a cell has been used by many investigators to reduce mRNA levels for a particular protein and then to study the effects (for general reviews see refs. 1 and 2). When RNAi-mediated changes are done on scale, the assays resemble genetic screens and allow many aspects of mammalian cell biology to be studied that were inaccessible previously.siRNA-based screens have been used to study many cell biological processes, including cell survival, induction of apoptosis, changes in endocytosis, and chromosome integrity (3-6). shRNAbased screens have examined NF-B signal transduction, proteasome function, p53 signal transduction, modulation of RAS oncogene activity, modulation of mitosis, and mammary cell transformation (7-12). Our current studies have focused on the differences in kinase requirements in various human cells. The kinase family was chosen for several reasons. Kinases are known to play key roles in many cell decision-making processes (13,14). Their biochemistry is well known, and although many kinases have been studied in detail, there are numerous family members that are essentially unstudied to date. Finally, many chemical screens and subsequent medicinal chemistry have shown that effective antikinase, small-molecule inhibitors can be found that have cell-specific effects. Some of these small-molecule inhibitors have been advanced as clinical candidates for drug development, and the first ...

Recently, the structure of the core complex was solved in the Metastatic lymph node 51 [MLN51 (also known as CASC3)] is a component of the exon junction complex (EJC), which is assembled on spliced mRNAs and plays important roles in post-splicing events. The four proteins of the EJC core, MLN51, MAGOH, Y14 and EIF4AIII shuttle between the cytoplasm and the nucleus. However, unlike the last three, MLN51 is mainly detected in the cytoplasm, suggesting that it plays an additional function in this compartment. In the present study, we show that MLN51 is recruited into cytoplasmic aggregates known as stress granules (SGs) together with the SG-resident proteins, fragile X mental retardation protein (FMRP), poly(A) binding protein (PABP) and poly(A) + RNA. MLN51 specifically associates with SGs via its C-terminal region, which is dispensable for its incorporation in the EJC. MLN51 does not promote SG formation but its silencing, or the overexpression of a mutant lacking its Cterminal region, alters SG assembly. Finally, in human breast carcinomas, MLN51 is sometimes present in cytoplasmic foci also positive for FMRP and PABP, suggesting that SGs formation occurs in malignant tumours.Supplementary material available online at

Metastatic Lymph Node 51 (MLN51) cDNA was isolated by differential screening of a human breast cancer metastasis cDNA library. MLN51 cDNA encodes a novel human protein of 703 residues that shares no significant homology to any known protein. However MLN51 is well conserved between vertebrate and invertebrate species suggesting an important biological function. The amino terminal half of the protein contains a coiled-coil domain and two potential nuclear localization signals (NLS). The carboxy terminal half contains one SH2 and four SH3 binding motifs. The coiled-coil domain promotes MLN51 oligomerization in transfected cells. When transiently expressed, the MLN51 protein is mainly found in the cytoplasm with a weak nuclear staining. However, deletion of the carboxy terminal half of the protein allows the targeting of the protein to the nucleus, demonstrating that the NLSs are functional. MLN51 is ubiquitously expressed in normal tissues. Human breast carcinomas show MLN51 overexpression in malignant epithelial cells. The uncommon association of protein -protein interaction domains often found either in nuclear or in cytoplasmic signaling proteins raises a possible nucleo-cytoplasmic function for MLN51.

The exon junction complex (EJC) allows the spliceosome to communicate with other cellular machinery. This study shows that assembled EJC cores are enriched in nuclear regions around speckles, called perispeckles. Speckles and perispeckles may represent specialized nuclear regions for messenger ribonucleoprotein maturation.

MLN64, is invariably coamplified and coexpressed with erbB-2 in breast cancers. The human MLN64 and ERBB2 genes are positioned at less than 50 kb from each other, on chromosome 17q12. To understand the molecular basis of MLN64 overexpression in cancer, the genomic region containing the MLN64 and ERBB2 genes was isolated and mapped. The two genes, DARPP32 and Telethonin, flanking MLN64 respectively on its centromeric and telomeric sides, although coamplified, are not overexpressed in breast cancer cells, indicating that gene amplification is not sufficient to allow overexpression. The MLN64 minimal promoter was isolated and found to be a housekeeping gene promoter containing four potential Sp1 binding elements. Using Sp1-deficient Drosophila SL2 cells, MLN64 promoter activity was induced in a dosedependent manner by exogenous Sp1 addition. Furthermore, mutation of each individual Sp1 element resulted in a significant decrease in reporter gene activity, indicating that all the Sp1 binding elements are functional and act together to promote gene expression. Since the ERBB2 promoter is also positively regulated by Sp1, this study indicates that MLN64 and ERBB2 genes share common transcriptional controls together with a physical link on chromosome 17q. We speculate that, in addition to the oncogenic potential of erbB-2 overexpression, the unbalanced action of MLN64 contributes to the poor clinical outcome of breast tumors bearing this amplified region.

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Despite the need for more effective drug treatments to address muscle atrophy and disease, physiologically accurate in vitro screening models and higher information content preclinical assays that aid in the discovery and development of novel therapies are lacking. To this end, MyoScreen was developed: a robust and versatile high-throughput high-content screening (HT/HCS) platform that integrates a physiologically and pharmacologically relevant micropatterned human primary skeletal muscle model with a panel of pertinent phenotypic and functional assays. MyoScreen myotubes form aligned, striated myofibers, and they show nerve-independent accumulation of acetylcholine receptors (AChRs), excitation-contraction coupling (ECC) properties characteristic of adult skeletal muscle and contraction in response to chemical stimulation. Reproducibility and sensitivity of the fully automated MyoScreen platform are highlighted in assays that quantitatively measure myogenesis, hypertrophy and atrophy, AChR clusterization, and intracellular calcium release dynamics, as well as integrating contractility data. A primary screen of 2560 compounds to identify stimulators of myofiber regeneration and repair, followed by further biological characterization of two hits, validates MyoScreen for the discovery and testing of novel therapeutics. MyoScreen is an improvement of current in vitro muscle models, enabling a more predictive screening strategy for preclinical selection of the most efficacious new chemical entities earlier in the discovery pipeline process.