Structure of Musashi1 in a complex with target RNA: the role of aromatic stacking interactions

Mammalian Musashi1 (Msi1) is an RNA-binding protein that regulates the translation of target mRNAs, and participates in the maintenance of cell 'stemness' and tumorigenesis. Msi1 reportedly binds to the 3'-untranslated region of mRNA of Numb, which encodes Notch inhibitor, and impedes initiation of its translation by competing with eIF4G for PABP binding, resulting in triggering of Notch signaling. Here, the mechanism by which Msi1 recognizes the target RNA sequence using its Ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2 has been revealed on identification of the minimal binding RNA for each RBD and determination of the three-dimensional structure of the RBD1:RNA complex. Unique interactions were found for the recognition of the target sequence by Msi1 RBD1: adenine is sandwiched by two phenylalanines and guanine is stacked on the tryptophan in the loop between β1 and α1. The minimal recognition sequences that we have defined for Msi1 RBD1 and RBD2 have actually been found in many Msi1 target mRNAs reported to date. The present study provides molecular clues for understanding the biology involving Musashi family proteins.     

3′-Untranslated region of doublecortin mRNA is a binding target of the Musashi1 RNA-binding protein

Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural stem cells, and is considered to be a stemness factor. A known function of Msi1 is translational repression of specifically bound mRNAs. Although the basic mechanism and some target RNAs have been reported, further survey of interactors is necessary to understand the integrated function of Msi1. By screening using an mRNA display technique, we found that doublecortin (dcx) mRNA is a specific binding target of Msi1 in vitro. We confirmed that Msil repressed translation of a luciferase reporter gene linked to the selected 3′-untranslated region fragment of dcx in Neuro2A cells.     

Neural RNA-binding protein Musashi1 inhibits translation initiation by competing with eIF4G for PABP

Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural stem cells. We previously reported that Msi1 contributes to the maintenance of the immature state and self-renewal activity of neural stem cells through translational repression of m-Numb. However, its translation repression mechanism has remained unclear. Here, we identify poly(A) binding protein (PABP) as an Msi1-binding protein, and find Msi1 competes with eIF4G for PABP binding. This competition inhibits translation initiation of Msi1's target mRNA. Indeed, deletion of the PABP-interacting domain in Msi1 abolishes its function. We demonstrate that Msi1 inhibits the assembly of the 80S, but not the 48S, ribosome complex. Consistent with these conclusions, Msi1 colocalizes with PABP and is recruited into stress granules, which contain the stalled preinitiation complex. However, Msi1 with mutations in two RNA recognition motifs fails to accumulate into stress granules. These results provide insight into the mechanism by which sequence-specific translational repression occurs in stem cells through the control of translation initiation.     

Musashi-2 Silencing Exerts Potent Activity against Acute Myeloid Leukemia and Enhances Chemosensitivity to Daunorubicin

RNA-binding protein Musashi-2 (Msi2) is known to play a critical role in leukemogenesis and contributes to poor clinical prognosis in acute myeloid leukemia (AML). However, the effect of Msi2 silencing on treatment for AML still remains poorly understood. In this study, we used lentivirus-mediated RNA interference targeting Msi2 to investigate the resulting changes in cellular processes and the underlying mechanisms in AML cell lines as well as primary AML cells isolated from AML patients. We found that Msi2 was highly expressed in AML cells, and its depletion inhibited Ki-67 expression and resulted in decreased in vitro and in vivo proliferation. Msi2 silencing induced cell cycle arrest in G0/G1 phase, with decreased Cyclin D1 and increased p21 expression. Msi2 silencing induced apoptosis through down-regulation of Bcl-2 expression and up-regulation of Bax expression. Suppression of Akt, Erk1/2 and p38 phosphorylation also contributed to apoptosis mediated by Msi2 silencing. Finally, Msi2 silencing in AML cells also enhanced their chemosensitivity to daunorubicin. Conclusively, our data suggest that Msi2 is a promising target for gene therapy to optimize conventional chemotherapeutics in AML treatment.     

Constitutive Musashi1 expression impairs mouse postnatal development and intestinal homeostasis

Evolutionarily conserved RNA-binding protein Musashi1 (Msi1) can regulate developmentally relevant genes. Here we report the generation and characterization of a mouse model that allows inducible Msi1 overexpression in a temporal and tissue-specific manner. We show that ubiquitous Msi1 induction in ∼5-wk-old mice delays overall growth, alters organ-to-body proportions, and causes premature death. Msi1-overexpressing mice had shortened intestines, diminished intestinal epithelial cell (IEC) proliferation, and decreased growth of small intestine villi and colon crypts. Although Lgr5-positive intestinal stem cell numbers remained constant in Msi1-overexpressing tissue, an observed reduction in Cdc20 expression provided a potential mechanism underlying the intestinal growth defects. We further demonstrated that Msi1 overexpression affects IEC differentiation in a region-specific manner, with ileum tissue being influenced the most. Ilea of mutant mice displayed increased expression of enterocyte markers, but reduced expression of the goblet cell marker Mucin2 and fewer Paneth cells. A higher hairy and enhancer of split 1:mouse atonal homolog 1 ratio in ilea from Msi1-overexpressing mice implicated Notch signaling in inducing enterocyte differentiation. Together, this work implicates Msi1 in mouse postnatal development of multiple organs, with Notch signaling alterations contributing to intestinal defects. This new mouse model will be a useful tool to further elucidate the role of Msi1 in other tissue settings.     

Musashi2 modulates K562 leukemic cell proliferation and apoptosis involving the MAPK pathway

The RNA-binding protein Musashi2 (Msi2) has been identified as a master regulator within a variety of stem cell populations via the regulation of translational gene expression. A recent study has suggested that Msi2 is strongly expressed in leukemic cells of acute myeloid leukemia patients, and elevated Msi2 is associated with poor prognosis. However, the potential role of Msi2 in leukemogenesis is still not well understood. Here, we investigated the effect of Msi2 knockdown on the biological properties of leukemic cells. High expression of Msi2 was found in K562 and KG-1a leukemic cell lines, and low expression was observed in the U937 cell line. We transduced K562 cells with two independent adenoviral shRNA vectors targeting Msi2 and confirmed knockdown of Msi2 at the mRNA and protein levels. Msi2 silencing inhibited cell growth and caused cell cycle arrest by increasing the expression of p21 and decreasing the expression of cyclin D1 and cdk2. In addition, knockdown of Msi2 promoted cellular apoptosis via the upregulation of Bax and downregulation of Bcl-2 expression. Furthermore, Msi2 knockdown resulted in the inactivation of the ERK/MAPK and p38/MAPK pathways, but no remarkable change in p-AKT was observed. These data provide evidence that Msi2 plays an important role in leukemogenesis involving the MAPK signaling pathway, which indicates that Msi2 may be a novel target for leukemia treatment.     

The sequence selectivity of KSRP explains its flexibility in the recognition of the RNA targets

K-homology (KH) splicing regulator protein (KSRP) is a multi-domain RNA-binding protein that regulates different steps of mRNA metabolism, from mRNA splicing to mRNA decay, interacting with a broad range of RNA sequences. To understand how KSRP recognizes its different RNA targets it is necessary to define the general rules of KSRP–RNA interaction. We describe here a complete scaffold-independent analysis of the RNA-binding potential of the four KH domains of KSRP. The analysis shows that KH3 binds to the RNA with a significantly higher affinity than the other domains and recognizes specifically a G-rich target. It also demonstrates that the other KH domains of KSRP display different sequence preferences explaining the broad range of targets recognized by the protein. Further, KSRP shows a strong negative selectivity for sequences containing several adjacent Cytosines limiting the target choice of KSRP within single-stranded RNA regions. The in-depth analysis of the RNA-binding potential of the KH domains of KSRP provides us with an understanding of the role of low sequence specificity domains in RNA recognition by multi-domain RNA-binding proteins.     

The oncogenic RNA-binding protein Musashi1 is regulated by HuR via mRNA translation and stability in glioblastoma cells

Musashi1 (Msi1) is an evolutionarily conserved RNA-binding protein (RBP) that has profound implications in cellular processes such as stem cell maintenance, nervous system development, and tumorigenesis. Msi1 is highly expressed in many cancers, including glioblastoma, whereas in normal tissues, its expression is restricted to stem cells. Unfortunately, the factors that modulate Msi1 expression and trigger high levels in tumors are largely unknown. The Msi1 mRNA has a long 3' untranslated region (UTR) containing several AU- and U-rich sequences. This type of sequence motif is often targeted by HuR, another important RBP known to be highly expressed in tumor tissue such as glioblastoma and to regulate a variety of cancer-related genes. In this report, we show an interaction between HuR and the Msi1 3'-UTR, resulting in a positive regulation of Msi1 expression. We show that HuR increased MSI1 mRNA stability and promoted its translation. We also present evidence that expression of HuR and Msi1 correlate positively in clinical glioblastoma samples. Finally, we show that inhibition of cell proliferation, increased apoptosis, and changes in cell-cycle profile as a result of silencing HuR are partially rescued when Msi1 is ectopically expressed. In summary, our results suggest that HuR is an important regulator of Msi1 in glioblastoma and that this regulation has important biological consequences during gliomagenesis.     

Numb modulates intestinal epithelial cells toward goblet cell phenotype by inhibiting the Notch signaling pathway

Numb was originally identified as an important cell fate determinant that is asymmetrically inherited during mitosis and controls the fate of sibling cells by inhibiting the Notch signaling pathway in neural tissue. The small intestinal epithelium originates from the division of stem cells that reside in the crypt, which further differentiate into goblet cells, absorptive cells, paneth cells, and enteroendocrine cells. However, Numb's involvement in the differentiation process of intestinal epithelium is largely unknown. In the present study, we confirm that both the Numb mRNA and protein isoforms are expressed in adult mouse intestinal mucosa. Numb protein is ubiquitously expressed throughout the crypt–villus axis of the small intestinal epithelium and is mainly localized to the cytoplasmic membrane. Down-regulation of endogenous Numb using RNA interference in cultured intestinal LS174T cells increased Notch signaling, leading to the up-regulation of Hes1 and the down-regulation of Hath1. Knockdown of Numb alleviated MUC2 protein expression and led to loss of the goblet cell phenotype in LS174Tl cells. Our results provide the first evidence that Numb, an important cell fate determinant, modulates intestinal epithelial cells towards the goblet cell phenotype by inhibiting the Notch signaling pathway.     

Beta-adrenergic agonists that down-regulate receptor mRNA up-regulate a M(r) 35,000 protein(s) that selectively binds to beta-adrenergic receptor mRNAs.

In an effort to explore the molecular basis for agonist-induced destabilization of beta-adrenergic receptor mRNA, we investigated the nature of RNA-binding proteins both in untreated and agonist-treated DDT1-MF2 smooth muscle cells. Messenger RNAs for the alpha 1b-, beta 1-, and beta 2-adrenergic receptors as well as for beta-globin were transcribed in vitro, incubated with cytosolic fractions, covalently cross-linked by short-wave UV light, and analyzed by SDS-polyacrylamide gel electrophoresis. A prominent M(r) 35,000 radiolabeled protein(s) with the following characteristics was identified: (i) binds selectively to beta 1- and beta 2-adrenergic receptor mRNAs, both of which undergo agonist-induced down-regulation; (ii) does not bind to either alpha 1b-adrenergic receptor mRNA, which does not undergo agonist induced down-regulation, or to beta-globin mRNA; (iii) displays binding to beta 2-adrenergic receptor mRNA that is selectively competed by poly(U) RNA, but not poly(A), -(C), or -(G) RNA; and (iv) displays binding to receptor mRNA that can be competed by RNA harboring destabilizer sequences that are AU-rich and AUUUA pentamer-rich. The abundance of the M(r) 35,000 RNA-binding protein selective for beta-adrenergic receptor message, a factor we term beta ARB protein, varies inversely with the level of receptor mRNA, being induced by agonists that down-regulate receptor mRNA.     

Musashi1: A stem cell marker no longer in search of a function

One of the earliest genes identified with stem and early progenitor cells is the RNA-binding protein, Musashi1 (Msi1). Through gene profiling of mammary epithelial cells transduced with Msi1, a unique autocrine signaling pathway was identified that activates both the Wnt and Notch pathways 1. This process was associated with increased secretion of the growth factor, PLF1 and inhibition of the secreted Wnt pathway inhibitor, DKK3. Identification of PLF1 as an effector of these pathways in the absence of the DKK3 tumor suppressor provides a new avenue for investigating differences between normal and malignant tissues, and potentially targeting tumor stem cells.     

Hes-1-targeting siRNA inhibits the maturation of murine myeloid-derived dendritic cells

Activation of Notch by Jagged-1 may plays a pivotal role in maturation of dendritic cells (DCs), but the mechanism has not been completely defined. In the present study, Hes-1 (Hairy/enhancer-of-split)-targeting siRNA was used to confirm a role of Jagged-1-Notch signaling pathway activation in maturation of murine bone marrow-derived DCs and to search for a target that plays a critical role. The results showed that compared with the control, lipopolysaccharide or Zymosan A groups, Jagged-1 (a soluble Jagged 1/Fc chimera protein) effectively increased expression of Hes-1 and Deltex-1 mRNA, which could be reversed by DAPT (2, 4-diamino-5-phenylthiazole), a specific inhibitor of the Notch signaling pathway. Hes-1-targeting siRNA could successfully down-regulate the endogenous Hes-1 expression in the DCs. Concurrently, a significant down-regulation of CD40, CD80, CD86 and MHC-II expressions on the surface of the DCs was found with the reduction of IL-12 yielded by the DCs. Our results demonstrate that Hes-1-targeting siRNA can inhibit the maturation of the DCs induced by Jagged-1, indicating Hes-1 may be an important target of Notch signaling mediating the maturation of DCs.     

Function of RNA-binding protein Musashi-1 in stem cells

Musashi is an evolutionarily conserved family of RNA-binding proteins that is preferentially expressed in the nervous system. The first member of the Musashi family was identified in Drosophila. This protein plays an essential role in regulating the asymmetric cell division of ectodermal precursor cells known as sensory organ precursor cells through the translational regulation of target mRNA. In the CNS of Drosophila larvae, however, Musashi is expressed in proliferating neuroblasts and likely has a different function. Its probable mammalian homologue, Musashi-1, is a neural RNA-binding protein that is strongly expressed in fetal and adult neural stem cells (NSCs). Mammalian Musashi-1 augments Notch signaling through the translational repression of its target mRNA, m-Numb, thereby contributing to the self-renewal of NSCs. In addition to its functions in NSCs, the role of mammalian Musashi-1 protein in epithelial stem cells, including intestinal and mammary gland stem cells, is attracting increasing interest.     

LncRNA NBR2 inhibits epithelial-mesenchymal transition by regulating Notch1 signaling in osteosarcoma cells

Long noncoding RNAs (lncRNAs) have been identified to have increasingly important roles in tumorigenesis, and they may serve as novel biomarkers for cancer therapy. Recent studies have demonstrated that lncRNA NBR2 (neighbor of BRCA1 gene 2), a novel identified lncRNA, is decreased in several cancers; however, the role of NBR2 in the development of osteosarcoma has not been elucidated. In our study, we found that NBR2 expression was downregulated in osteosarcoma tissues, and osteosarcoma cases with lower NBR2 expression exhibited a shorter overall survival time compared with those with higher NBR2 expression. NBR2 overexpression inhibited osteosarcoma cell proliferation, invasion, and migration but did not increase apoptosis. Furthermore, RNA-binding protein immunoprecipitation assays confirmed that NBR2 directly binds to Notch1 protein. Furthermore, overexpression of Notch1 in NBR2-overexpressing osteosarcoma cells reversed the effects of NBR2 on cell proliferation, invasion, migration, and epithelial-mesenchymal transition. The in vivo results showed that NBR2 overexpression inhibited tumor growth in nude mice that were inoculated with osteosarcoma cells. NBR2 overexpression also suppressed the messenger RNA (mRNA) expression of Notch1, N-cadherin, and vimentin and increased the mRNA expression of E-cadherin in the tumor tissues. These data indicated that NBR2 served as a tumor suppressor gene in osteosarcoma and inhibited osteosarcoma cell proliferation, invasion, and migration. The current study provides a novel insight and treatment strategy for osteosarcoma.     

Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1

In C. elegans, the Notch receptor GLP-1 is localized within the germline and early embryo by translational control of glp-1 mRNA. RNA elements in the glp-1 3'untranslated region (3' UTR) are necessary for repression of glp-1 translation in germ cells, and for localization of translation to anterior cells of the early embryo. The direct regulators of glp-1 mRNA are not known. Here, we show that a 34 nucleotide region of the glp-1 3' UTR contains two regulatory elements, an element that represses translation in germ cells and posterior cells of the early embryo, and an element that inhibits repressor activity to promote translation in the embryo. Furthermore, we show that the STAR/KH domain protein GLD-1 binds directly and specifically to the repressor element. Depletion of GLD-1 activity by RNA interference causes loss of endogenous glp-1 mRNA repression in early meiotic germ cells, and in posterior cells of the early embryo. Therefore, GLD-1 is a direct repressor of glp-1 translation at two developmental stages. These results suggest a new function for GLD-1 in regulating early embryonic asymmetry. Furthermore, these observations indicate that precise control of GLD-1 activity by other regulatory factors is important to localize this Notch receptor, and contributes to the spatial organization of Notch signaling.