Suzanne C. Burns

Sequence signatures and mRNA concentration can explain two-thirds of protein abundance variation in a human cell line

Transcription, mRNA decay, translation and protein degradation are essential processes during eukaryotic gene expression, but their relative global contributions to steady‐state protein concentrations in multi‐cellular eukaryotes are largely unknown. Using measurements of absolute protein and mRNA abundances in cellular lysate from the human Daoy medulloblastoma cell line, we quantitatively evaluate the impact of mRNA concentration and sequence features implicated in translation and protein degradation on protein expression. Sequence features related to translation and protein degradation have an impact similar to that of mRNA abundance, and their combined contribution explains two‐thirds of protein abundance variation. mRNA sequence lengths, amino‐acid properties, upstream open reading frames and secondary structures in the 5′ untranslated region (UTR) were the strongest individual correlates of protein concentrations. In a combined model, characteristics of the coding region and the 3′UTR explained a larger proportion of protein abundance variation than characteristics of the 5′UTR. The absolute protein and mRNA concentration measurements for >1000 human genes described here represent one of the largest datasets currently available, and reveal both general trends and specific examples of post‐transcriptional regulation.

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.

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.

Genomic Analyses of Musashi1 Downstream Targets Show a Strong Association with Cancer-related Processes

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.

Genomic Analyses of the RNA-binding Protein Hu Antigen R (HuR) Identify a Complex Network of Target Genes and Novel Characteristics of Its Binding Sites

Background: The RNA-binding protein HuR is involved in a range of cellular processes and several diseases. Results: We reveal the characteristics of HuR binding using genomic methods and explore its network of targets. Conclusion: Our results reveal the complexity of RBP binding, corroborate the concept of post-transcriptional networks and suggest an interplay between miRNAs and RBPs. Significance: An understanding of HuR informs our knowledge of RBPs and may lead to effective treatments for related diseases. The ubiquitously expressed RNA-binding protein Hu antigen R (HuR) or ELAVL1 is implicated in a variety of biological processes as well as being linked with a number of diseases, including cancer. Despite a great deal of prior investigation into HuR, there is still much to learn about its function. We take an important step in this direction by conducting cross-linking and immunoprecipitation and RNA sequencing experiments followed by an extensive computational analysis to determine the characteristics of the HuR binding site and impact on the transcriptome. We reveal that HuR targets predominantly uracil-rich single-stranded stretches of varying size, with a strong conservation of structure and sequence composition. Despite the fact that HuR sites are observed in intronic regions, our data do not support a role for HuR in regulating splicing. HuR sites in 3′-UTRs overlap extensively with predicted microRNA target sites, suggesting interplay between the functions of HuR and microRNAs. Network analysis showed that identified targets containing HuR binding sites in the 3′ UTR are highly interconnected.

Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy.

BackgroundMusashi1 (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.MethodsWe used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells.ResultsWe observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy.ConclusionOur data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy.

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.

Genomic Analyses Reveal Broad Impact of miR-137 on Genes Associated with Malignant Transformation and Neuronal Differentiation in Glioblastoma Cells

miR-137 plays critical roles in the nervous system and tumor development; an increase in its expression is required for neuronal differentiation while its reduction is implicated in gliomagenesis. To evaluate the potential of miR-137 in glioblastoma therapy, we conducted genome-wide target mapping in glioblastoma cells by measuring the level of association between PABP and mRNAs in cells transfected with miR-137 mimics vs. controls via RIPSeq. Impact on mRNA levels was also measured by RNASeq. By combining the results of both experimental approaches, 1468 genes were found to be negatively impacted by miR-137 – among them, 595 (40%) contain miR-137 predicted sites. The most relevant targets include oncogenic proteins and key players in neurogenesis like c-KIT, YBX1, AKT2, CDC42, CDK6 and TGFβ2. Interestingly, we observed that several identified miR-137 targets are also predicted to be regulated by miR-124, miR-128 and miR-7, which are equally implicated in neuronal differentiation and gliomagenesis. We suggest that the concomitant increase of these four miRNAs in neuronal stem cells or their repression in tumor cells could produce a robust regulatory effect with major consequences to neuronal differentiation and tumorigenesis.

IGF2BP3 modulates the interaction of invasion-associated transcripts with RISC

Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) expression correlates with malignancy, but its role(s) in pathogenesis remains enigmatic. We interrogated the IGF2BP3-RNA interaction network in pancreatic ductal adenocarcinoma (PDAC) cells. Using a combination of genome-wide approaches, we have identified 164 direct mRNA targets of IGF2BP3. These transcripts encode proteins enriched for functions such as cell migration, proliferation, and adhesion. Loss of IGF2BP3 reduced PDAC cell invasiveness and remodeled focal adhesion junctions. Individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) revealed significant overlap of IGF2BP3 and microRNA (miRNA) binding sites. IGF2BP3 promotes association of the RNA-induced silencing complex (RISC) with specific transcripts. Our results show that IGF2BP3 influences a malignancy-associated RNA regulon by modulating miRNA-mRNA interactions.