PARalyzer: definition of RNA binding sites from PAR-CLIP short-read sequence data

Crosslinking and immunoprecipitation (CLIP) protocols have made it possible to identify transcriptome-wide RNA-protein interaction sites. In particular, PAR-CLIP utilizes a photoactivatable nucleoside for more efficient crosslinking. We present an approach, centered on the novel PARalyzer tool, for mapping high-confidence sites from PAR-CLIP deep-sequencing data. We show that PARalyzer delineates sites with a high signal-to-noise ratio. Motif finding identifies the sequence preferences of RNA-binding proteins, as well as seed-matches for highly expressed microRNAs when profiling Argonaute proteins. Our study describes tailored analytical methods and provides guidelines for future efforts to utilize high-throughput sequencing in RNA biology. PARalyzer is available at http://www.genome.duke.edu/labs/ohler/research/PARalyzer/

A STAT3 Gene Expression Signature in Gliomas is Associated with a Poor Prognosis

Gliomas frequently display constitutive activation of the transcription factor STAT3, a protein that is known to be able to mediate neoplastic transformation. STAT3 regulates genes that play a central role in cellular survival, proliferation, self-renewal, and invasion, and a cohort of STAT3 target genes have been found that are commonly coexpressed in human cancers. Thus, these genes likely subserve the transforming ability of constitutively activated STAT3. To determine whether the coordinated expression of STAT3 target genes is present in a subset of human gliomas, and whether this changes the biology of these tumors in patients, gene expression analysis was performed in four distinct human glioma data sets for which patient survival information was available. Coordinate expression of STAT3 targets was significantly associated with poor patient outcome in each data set. Specifically, patients with tumors displaying high expression of STAT3 targets had a shorter median survival time compared to patients whose tumors had low expression of STAT3 targets. These data suggest that constitutively activated STAT3 in gliomas can alter the biology of these tumors, and that development of targeted STAT3 inhibitors would likely be of particular benefit in treatment of this disease

Tissue Type-Specific Expression of the dsRNA-Binding Protein 76 and Genome-Wide Elucidation of Its Target mRNAs

Background: RNA-binding proteins accompany all steps in the life of mRNAs and provide dynamic gene regulatory functions for rapid adjustment to changing extra-or intracellular conditions. The association of RNA-binding proteins with their targets is regulated through changing subcellular distribution, post-translational modification or association with other proteins. Methodology: We demonstrate that the dsRNA binding protein 76 (DRBP76), synonymous with nuclear factor 90, displays inherently distinct tissue type-specific subcellular distribution in the normal human central nervous system and in malignant brain tumors of glial origin. Altered subcellular localization and isoform distribution in malignant glioma indicate that tumor-specific changes in DRBP76-related gene products and their regulatory functions may contribute to the formation and/or maintenance of these tumors. To identify endogenous mRNA targets of DRBP76, we performed RNA-immunoprecipitation and genome-wide microarray analyses in HEK293 cells, and identified specific classes of transcripts encoding critical functions in cellular metabolism. Significance: Our data suggest that physiologic DRBP76 expression, isoform distribution and subcellular localization are profoundly altered upon malignant transformation. Thus, the functional role of DRBP76 in co- or post-transcriptional gene regulation may contribute to the neoplastic phenotype

Differences in RNA metabolism between human coding and non-coding RNA

Non UBCUnreviewedAuthor affiliation: Berlin Institute for Medical Systems BiologyPostdoctora

Ribonomic Analysis of Human Pum1 Reveals cis-trans Conservation across Species despite Evolution of Diverse mRNA Target Sets▿ †

PUF family proteins are among the best-characterized regulatory RNA-binding proteins in nonmammalian species, but relatively little is known about mRNA targets or functions of mammalian PUF proteins. In this study, we used ribonomic analysis to identify and analyze mRNAs associated with ribonucleoproteins containing an endogenous human PUF protein, Pum1. Pum1-associated mRNAs were highly enriched for genes encoding proteins that function in transcriptional regulation and cell cycle/proliferation, results consistent with the posttranscriptional RNA regulon model and the proposed ancestral functions of PUF proteins in stem cell biology. Analysis of 3′ untranslated region sequences of Pum1-associated mRNAs revealed a core Pum1 consensus sequence, UGUAHAUA. Pum1 knockdown demonstrated that Pum1 enhances decay of associated mRNAs, and relocalization of Pum1 to stress granules suggested that Pum1 functions in repression of translation. This study is the first in vivo genome-wide mRNA target identification of a mammalian PUF protein and provides direct evidence that human PUF proteins regulate stability of associated mRNAs. Comparison of Pum1-associated mRNAs to mRNA targets of PUF proteins from Saccharomyces cerevisiae and Drosophila melanogaster demonstrates how a well-conserved RNA-binding domain and cognate binding sequence have been evolutionarily rewired to regulate the collective expression of different sets of functionally related genes

A STAT3 Gene Expression Signature in Gliomas is Associated with a Poor Prognosis.

Gliomas frequently display constitutive activation of the transcription factor STAT3, a protein that is known to be able to mediate neoplastic transformation. STAT3 regulates genes that play a central role in cellular survival, proliferation, self-renewal, and invasion, and a cohort of STAT3 target genes have been found that are commonly coexpressed in human cancers. Thus, these genes likely subserve the transforming ability of constitutively activated STAT3. To determine whether the coordinated expression of STAT3 target genes is present in a subset of human gliomas, and whether this changes the biology of these tumors in patients, gene expression analysis was performed in four distinct human glioma data sets for which patient survival information was available. Coordinate expression of STAT3 targets was significantly associated with poor patient outcome in each data set. Specifically, patients with tumors displaying high expression of STAT3 targets had a shorter median survival time compared to patients whose tumors had low expression of STAT3 targets. These data suggest that constitutively activated STAT3 in gliomas can alter the biology of these tumors, and that development of targeted STAT3 inhibitors would likely be of particular benefit in treatment of this disease

Transcriptomic Response Dynamics of Human Primary and Immortalized Adrenocortical Cells to Steroidogenic Stimuli

Adrenal steroid hormone production is a dynamic process stimulated by adrenocorticotropic hormone (ACTH) and angiotensin II (AngII). These ligands initialize a rapid and robust gene expression response required for steroidogenesis. Here, we compare the predominant human immortalized cell line model, H295R cell, with primary cultures of adult adrenocortical cells derived from human kidney donors. We performed temporally resolved RNA-seq on primary cells stimulated with either ACTH or AngII at multiple time points. The magnitude of the expression dynamics elicited by ACTH was greater than AngII in primary cells. This is likely due to the larger population of adrenocortical cells that are responsive to ACTH. The dynamics of stimulus-induced expression in H295R cells are mostly recapitulated in primary cells. However, there are some expression responses in primary cells absent in H295R cells. These data are a resource for the endocrine community and will help researchers determine whether H295R is an appropriate model for the specific aspect of steroidogenesis that they are studying

Radiation enhances the invasive potential of primary glioblastoma cells via activation of the Rho signaling pathway.

Glioblastoma multiforme (GBM) is among the most treatment-refractory of all human tumors. Radiation is effective at prolonging survival of GBM patients; however, the vast majority of GBM patients demonstrate progression at or near the site of original treatment. We have identified primary GBM cell lines that demonstrate increased invasive potential upon radiation exposure. As this represents a novel mechanism by which radiation-treated GBMs can fail therapy, we further investigated the identity of downstream signaling molecules that enhance the invasive phenotype of irradiated GBMs. Matrigel matrices were used to compare the extent of invasion of irradiated vs. non-irradiated GBM cell lines UN3 and GM2. The in vitro invasive potential of these irradiated cells were characterized in the presence of both pharmacologic and dominant negative inhibitors of extracellular matrix and cell signaling molecules including MMP, uPA, IGFR, EGFR, PI-3K, AKT, and Rho kinase. The effect of radiation on the expression of these signaling molecules was determined with Western blot assays. Ultimately, the in vitro tumor invasion results were confirmed using an in vivo 9L GBM model in rats. Using the primary GBM cell lines UN3 and GM2, we found that radiation enhances the invasive potential of these cells via activation of EGFR and IGFR1. Our findings suggest that activation of Rho signaling via PI-3K is required for radiation-induced invasion, although not required for invasion under physiologic conditions. This report clearly demonstrates that radiation-mediated invasion is fundamentally distinct from invasion under normal cellular physiology and identifies potential therapeutic targets to overcome this phenomenon