Musashi1 (Msi1) is a highly conserved RNA-binding protein with pivotal functions in stem cell maintenance, nervous system development, and tumorigenesis. Despite its importance, only three direct mRNA targets have been characterized so far: m-numb, CDKN1A, and c-mos. Msi1 has been shown to affect their translation by binding to short elements located in the 3-untranslated region. To better understand Msi1 functions, we initially performed an RIP-Chip analysis in HEK293T cells; this method consists of isolation of specific RNA-protein complexes followed by identification of the RNA component via microarrays. A group of 64 mRNAs was found to be enriched in the Msi1-associated population compared with controls. These genes belong to two main functional categories pertinent to tumorigenesis: 1) cell cycle, cell proliferation, cell differentiation, and apoptosis and 2) protein modification (including ubiquitination and ubiquitin cycle). To corroborate our findings, we examined the impact of Msi1 expression on both mRNA (transcriptomic) and protein (proteomic) expression levels. Genes whose mRNA levels were affected by Msi1 expression have a Gene Ontology distribution similar to RIP-Chip results, reinforcing Msi1 participation in cancer-related processes. The proteomics study revealed that Msi1 can have either positive or negative effects on gene expression of its direct targets. In summary, our results indicate that Msi1 affects a network of genes and could function as a master regulator during development and tumor formation.

Background: Musashi1 (Msi1) is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer.

Stenotrophomonas maltophilia is an opportunistic pathogen characterized for its intrinsic low susceptibility to several antibiotics. Part of this low susceptibility relies on the expression of chromosomally encoded multidrug efflux pumps, with SmeDEF being the most relevant antibiotic resistance efflux pump so far studied in this bacterial species. Expression of smeDEF is downregulated by the SmeT repressor, encoded upstream smeDEF, in its complementary DNA strand. In the present article we present the crystal structure of SmeT and analyze its interactions with its cognate operator. Like other members of the TetR family of transcriptional repressors, SmeT behaves as a dimer and presents some common structural features with other TetR proteins like TtgR, QacR, and TetR. Differing from other TetR proteins for which the structure is available, SmeT turned out to have two extensions at the N and C termini that might be relevant for its function. Besides, SmeT presents the smallest binding pocket so far described in the TetR family of transcriptional repressors, which may correlate with a specific type and range of effectors. In vitro studies revealed that SmeT binds to a 28-bp pseudopalindromic region, forming two complexes. This operator region was found to overlap the promoters of smeT and smeDEF. This finding is consistent with a role for SmeT simultaneously down-regulating smeT and smeDEF transcription, likely by steric hindrance on RNA polymerase binding to DNA.Opportunistic pathogens intrinsically resistant to antibiotics are currently a relevant health problem (1). Although several elements contribute to the intrinsic resistance of these bacteria (2, 3), the active efflux of antibiotics is a common mechanism relevant for their phenotype. Antibiotic efflux is because of the activity of multidrug (MDR) 5 efflux pumps (4 -7). Those elements are universally distributed among all living systems (8). In Gram-negative bacteria, the best characterized MDR pumps belong to the resistance nodulation division family. In most cases MDR pumps are down-regulated by specific transcription factors located upstream of the operon coding for the pump (9). Stable expression and, thus, a higher level of resistance is achieved by mutations in these regulatory elements. It is worth noting here that the constitutive expression of MDR pumps in these mutants is frequently linked to fitness costs (10 -12). This indicates the need of a precise and stringent regulation of the system to avoid the nonspecific expression of these elements.Stenotrophomonas maltophilia is an opportunistic pathogen that is considered as a prototype of intrinsically resistant bacteria (13). The first MDR pump described in this bacterial species was SmeDEF (14 -16), an MDR determinant that contributes to intrinsic (17) and acquired (14, 16) resistance to several antibiotics and to biocides (18) in S. maltophilia. Expression of smeDEF is down-regulated by the transcriptional repressor SmeT (19,20), located upstream, and in the complementary strand of smeDEF. Mut...

BackgroundmicroRNAs (miRNAs) have been implicated in the control of many biological processes and their deregulation has been associated with many cancers. In recent years, the cancer stem cell (CSC) concept has been applied to many cancers including pediatric. We hypothesized that a common signature of deregulated miRNAs in the CSCs fraction may explain the disrupted signaling pathways in CSCs.Methodology/ResultsUsing a high throughput qPCR approach we identified 26 CSC associated differentially expressed miRNAs (DEmiRs). Using BCmicrO algorithm 865 potential CSC associated DEmiR targets were obtained. These potential targets were subjected to KEGG, Biocarta and Gene Ontology pathway and biological processes analysis. Four annotated pathways were enriched: cell cycle, cell proliferation, p53 and TGF-beta/BMP. Knocking down hsa-miR-21-5p, hsa-miR-181c-5p and hsa-miR-135b-5p using antisense oligonucleotides and small interfering RNA in cell lines led to the depletion of the CSC fraction and impairment of sphere formation (CSC surrogate assays).ConclusionOur findings indicated that CSC associated DEmiRs and the putative pathways they regulate may have potential therapeutic applications in pediatric cancers.