Mei Qiao

A compendium of RNA-binding motifs for decoding gene regulation

RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.

Smurf1 Inhibits Osteoblast Differentiation and Bone Formation in Vitro and in Vivo

Bone morphogenetic proteins (BMPs) are required for normal postnatal bone formation and osteoblast differentiation. There is evidence from recent studies that BMP signaling in osteoblasts is controlled by an ubiquitin-proteasome regulatory mechanism involving a cascade of enzymatic reactions. The specificity of protein ubiquitination is determined by E3 ubiquitin ligases, which play a crucial role in defining substrate specificity and subsequent protein degradation by 26S proteasomes. We have examined the role of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1), a member of the Hect domain family of E3 ubiquitin ligases in osteoblast function. Smurf1 has been found to interact with BMP-activated Smad1 and -5 and to mediate degradation of these Smad proteins. Recently we have found that Smurf1 mediates the protein degradation of the osteoblast-specific transcription factor Runx2/Cbfa1. To determine the role of Smurf1 in osteoblast differentiation, in the present studies we transfected a Smurf1 expression plasmid into 2T3 osteoblast precursor cells and found that Smurf1 overexpression inhibits BMP signaling and osteoblast differentiation. To further investigate the role of Smurf1 in bone formation in vivo, we generated transgenic mice in which expression of the epitope-tagged Smurf1 transgene was targeted to osteoblasts using the murine 2.3-kb osteoblast-specific type I collagen promoter. In these transgenic mice, bone formation was significantly reduced during postnatal life. Our results demonstrate for the first time that Smurf1 plays a specific role in osteoblast differentiation and bone formation in vivo.

Site identification in high-throughput RNA–protein interaction data

MOTIVATION Post-transcriptional and co-transcriptional regulation is a crucial link between genotype and phenotype. The central players are the RNA-binding proteins, and experimental technologies [such as cross-linking with immunoprecipitation- (CLIP-) and RIP-seq] for probing their activities have advanced rapidly over the course of the past decade. Statistically robust, flexible computational methods for binding site identification from high-throughput immunoprecipitation assays are largely lacking however. RESULTS We introduce a method for site identification which provides four key advantages over previous methods: (i) it can be applied on all variations of CLIP and RIP-seq technologies, (ii) it accurately models the underlying read-count distributions, (iii) it allows external covariates, such as transcript abundance (which we demonstrate is highly correlated with read count) to inform the site identification process and (iv) it allows for direct comparison of site usage across cell types or conditions. AVAILABILITY AND IMPLEMENTATION We have implemented our method in a software tool called Piranha. Source code and binaries, licensed under the GNU General Public License (version 3) are freely available for download from http://smithlab.usc.edu. CONTACT andrewds@usc.edu SUPPLEMENTARY INFORMATION Supplementary data available at Bioinformatics online.

The zinc finger transcription factor Gli2 mediates bone morphogenetic protein 2 expression in osteoblasts in response to hedgehog signaling.

ABSTRACT Bone morphogenetic protein 2 (BMP-2) plays a critical role in osteoblast function. In Drosophila, Cubitus interruptus (Ci), which mediates hedgehog signaling, regulates gene expression of dpp, the ortholog of mammalian BMP-2. Null mutation of the transcription factor Gli2, a mammalian homolog of Ci, results in severe skeletal abnormalities in mice. We hypothesize that Gli2 regulates BMP-2 gene transcription and thus osteoblast differentiation. In the present study, we show that overexpression of Gli2 enhances BMP-2 promoter activity and mRNA expression in osteoblast precursor cells. In contrast, knocking down Gli2 expression by Gli2 small interfering RNA or genetic ablation of the Gli2 gene results in significant inhibition of BMP-2 gene expression in osteoblasts. Promoter analyses, including chromatin immunoprecipitation and electrophoretic mobility shift assays, provided direct evidence that Gli2 physically interacts with the BMP-2 promoter. Functional studies showed that Gli2 is required for osteoblast maturation in a BMP-2-dependent manner. Finally, Sonic hedgehog (Shh) stimulates BMP-2 promoter activity and osteoblast differentiation, and the effects of Shh are mediated by Gli2. Taken together, these results indicate that Gli2 mediates hedgehog signaling in osteoblasts and is a powerful activator of BMP-2 gene expression, which is required in turn for normal osteoblast differentiation.

Before It Gets Started: Regulating Translation at the 5′ UTR

Translation regulation plays important roles in both normal physiological conditions and diseases states. This regulation requires cis-regulatory elements located mostly in 5′ and 3′ UTRs and trans-regulatory factors (e.g., RNA binding proteins (RBPs)) which recognize specific RNA features and interact with the translation machinery to modulate its activity. In this paper, we discuss important aspects of 5′ UTR-mediated regulation by providing an overview of the characteristics and the function of the main elements present in this region, like uORF (upstream open reading frame), secondary structures, and RBPs binding motifs and different mechanisms of translation regulation and the impact they have on gene expression and human health when deregulated.

Matrix vesicles produced by osteoblast-like cells in culture become significantly enriched in proteoglycan-degrading metalloproteinases after addition of β-glycerophosphate and ascorbic acid

Matrix vesicles, media vesicles, and plasma membranes from three well-characterized, osteoblast-like cells (ROS 17/2.8, MG-63, and MC-3T3-E1) were evaluated for their content of enzymes capable of processing the extracellular matrix. Matrix vesicles were enriched in alkaline phosphatase specific activity over the plasma membrane and contained fully active neutral, but not acid, metalloproteinases capable of digesting proteoglycans, potential inhibitors of matrix calcification. Matrix vesicle enrichment in neutral metalloproteinase varied with the cell line, whereas collagenase, lysozyme, hyaluronidase, and tissue inhibitor of metalloproteinases (TIMP) were not found in any of the membrane fractions examined. MC-3T3-E1 cells were cultured for 32 days in the presence of ascorbic acid (100 μg/ml), β-glycerophosphate (5 mM), or a combination of the two, to assess changes in matrix vesicle enzymes during calcification. Ascorbate or β-glycerophosphate alone had no effect, but in combination produced significant increases in both active and total neutral metalloproteinase in matrix vesicles and plasma membranes, with the change seen in matrix vesicles being the most dramatic. This correlated with an increase in the formation of von Kossa-positive nodules. The results of the present study indicate that osteoblast-like cells produce matrix vesicles enriched in proteoglycan-degrading metalloproteinases. In addition, the observation that matrix vesicles contain significantly increased metalloproteinases under conditions favorable for mineralization in vitro lends support to the hypothesis that matrix vesicles play an important role in extracellular matrix processing and calcification in bone.

The oncogenic RNA-binding protein Musashi1 is regulated by tumor suppressor miRNAs

Musashi1 (Msi1) is an evolutionarily conserved RNA-binding protein that has been implicated in processes like stem cell fate, nervous system development, and tumorigenesis via its activities as a specific regulator of translation. While Msi1 is barely detected in normal adult tissue, it has been observed to be highly expressed in numerous tumor types (e.g. breast, colon, medulloblastoma, glioblastoma, and et cetera). Unfortunately, the molecular cues that are responsible for Msi1 upregulation in cancer cells are largely unknown. Tumor suppressor microRNAs (miRNAs) are known for targeting genes with oncogenic properties like Msi1 and for being either downregulated or deleted in tumor tissue. We observed that Msi1 long 3’UTR region is potentially targeted by several tumor suppressor miRNAs (miR-34a, -101, -128, -137, and -138). Western blotting of endogenous Msi1 protein as well as luciferase assays confirmed Msi1 regulation by these tumor suppressor miRNAs. Furthermore, we observed when examining different cellular states that these miRNAs and Msi1 have opposite expression profiles. Cell proliferation inhibition induced by the tumor suppressor miRNAs was partially rescued by Msi1 transgenic expression. We conclude that tumor suppressor miRNAs are direct and influential regulators of Msi1, affecting its expression pattern during tumorigenesis of malignant nervous system tumors.

The RNA-Binding Protein Musashi1 Affects Medulloblastoma Growth via a Network of Cancer-Related Genes and Is an Indicator of Poor Prognosis

Musashi1 (Msi1) is a highly conserved RNA-binding protein that is required during the development of the nervous system. Msi1 has been characterized as a stem cell marker, controlling the balance between self-renewal and differentiation, and has also been implicated in tumorigenesis, being highly expressed in multiple tumor types. We analyzed Msi1 expression in a large cohort of medulloblastoma samples and found that Msi1 is highly expressed in tumor tissue compared with normal cerebellum. Notably, high Msi1 expression levels proved to be a sign of poor prognosis. Msi1 expression was determined to be particularly high in molecular subgroups 3 and 4 of medulloblastoma. We determined that Msi1 is required for tumorigenesis because inhibition of Msi1 expression by small-interfering RNAs reduced the growth of Daoy medulloblastoma cells in xenografts. To characterize the participation of Msi1 in medulloblastoma, we conducted different high-throughput analyses. Ribonucleoprotein immunoprecipitation followed by microarray analysis (RIP-chip) was used to identify mRNA species preferentially associated with Msi1 protein in Daoy cells. We also used cluster analysis to identify genes with similar or opposite expression patterns to Msi1 in our medulloblastoma cohort. A network study identified RAC1, CTGF, SDCBP, SRC, PRL, and SHC1 as major nodes of an Msi1-associated network. Our results suggest that Msi1 functions as a regulator of multiple processes in medulloblastoma formation and could become an important therapeutic target.

Mice lacking 5-lipoxygenase have increased cortical bone thickness.

5-lipoxygenase (5-LO) metabolites have complex effects on bone cells. We have found that the leukotrine LTB4 and the peptido leukotrienes stimulate osteoclast formation in vitro and in vivo, and are likely involved in the generation of active osteoclast-like cells in giant cell tumors of bone. Recent preliminary observation in mice lacking the gene for 5-LO have not as yet demonstrated any effect on osteoclast function, but these mice have increased cortical bone, suggesting that 5-LO is a negative regulator of bone formation. 5-LO metabolites thus have unique effects on bone cell function, and clarifying their role may be important for the understanding of physiological as well as pathological bone remodeling.

Avian osteoclast cells are stimulated to resorb calcified matrices by and possess receptors for leukotriene B4

Abstract. Leukotriene B4 (LTB4) is elevated in inflammatory conditions and appears to be a potential mediator of inflammation. We have recently shown that this 5-lipoxygenase metabolite of arachidonic acid stimulates bone resorption in vitro and in vivo. In order to determine the mechanism whereby LTB4 causes bone resorption, avian osteoclasts were examined for the effects of LTB4 and for the presence of LTB4 receptors. Isolated avian osteoclast mononuclear precursor cells, which fuse in culture to form multinucleated cells, were chosen for receptor binding studies because this population is a morphologically similar source of osteoclasts, and large numbers of these cells can be obtained from egg-laying hens. Binding of LTB4 and activation would support the hypothesis of a direct effect of this compound on osteoclasts. LTB4 stimulated isolated avian osteoclasts to form resorption lacunae on calcified matrices and to increase their content of tartrate-resistant acid phosphatase (TRAP), a marker of activated osteoclasts. Receptor binding studies were performed at day 1, when the cells were mononuclear, at day 4, when mononuclear precursors were actively fusing, and at day 7, when fusion has slowed. Scatchard analysis of saturation binding data showed two classes of binding sites, a high- and low-affinity binding site with dissociation constants (KD) of 0.2–0.4 nM and 5.6–24 nM. Association studies showed rapid binding of LTB4 to the cells within 10 minutes. These data show that LTB4 accelerates fusion and activates highly enriched populations of avian osteoclasts and that LTB4 receptors are present in this cell population.