Scott A. Tenenbaum

Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome.

Fragile X syndrome results from the absence of the RNA binding FMR protein. Here, mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. We identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. We conclude that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and we identify candidate genes relevant to this phenotype.

Eukaryotic mRNPs may represent posttranscriptional operons

Genomic array analysis of endogenous mammalian ribonucleoproteins has recently revealed three novel findings: (1) mRNA binding proteins are associated with unique subpopulations of messages, (2) the compositions of these mRNA subsets can vary with growth conditions, and (3) the same mRNA species can be found in multiple mRNP complexes. Based on these and other findings, we propose a model of posttranscriptional gene expression in which mRNA binding proteins regulate mRNAs as subpopulations during cell growth and development. This model predicts that functionally related genes are regulated posttranscriptionally as groups by specific mRNA binding proteins that recognize sequence elements in common among the mRNAs.

Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays

Although in vitro methods have been used to identify putative targets of mRNA-binding proteins, direct in vivo methods are needed to identify endogenously associated mRNAs and their cognate proteins. Therefore, we have developed high-throughput methods to identify structurally and/or functionally related mRNA transcripts through their endogenous association with RNA-binding proteins. We have termed the identification and analysis of mRNA subsets using RNA-associated proteins ribonomics, and have established four primary steps for the method: (1) isolation of endogenous mRNA-protein complexes (mRNPs) under optimized conditions, (2) the en masse characterization of the protein and mRNA components associated with the targeted mRNP complexes, (3) identification of sequences or structural similarities among members of the mRNA subset, and (4) determination of functional relationships among the protein products coded for by members of the mRNA subset. We have hypothesized that mRNAs are organized into structurally and functionally linked groups to better affect information transfer through coordinate gene expression. The functional consequences of such organization would be to facilitate the production of proteins that regulate processes necessary for growth and differentiation. This article describes a series of biochemical techniques that deal with the first two steps of ribonomic profiling: purifying endogenous mRNP complexes and identifying multiple mRNA targets using microarray analysis.

Genome-wide Analysis Identifies Interleukin-10 mRNA as Target of Tristetraprolin

Tristetraprolin (TTP) is an RNA-binding protein required for the rapid degradation of mRNAs containing AU-rich elements. Targets regulated by TTP include the mRNAs encoding tumor necrosis factor-α, granulocyte-macrophage colony-stimulating factor, interleukin-2 (IL-2), and immediate early response 3. To identify novel target mRNAs of TTP in macrophages, we used a genome-wide approach that combines RNA immunoprecipitation and microarray analysis. A list was compiled of 137 mRNAs that are associated with TTP with an estimated accuracy on the order of 90%. Sequence analysis revealed a highly significant enrichment of AU-rich element motifs, with AUUUA pentamers present in 96% and UUAUUUAUU nonamers present in 44% of TTP-associated mRNAs. We further show that IL-10 is a novel target regulated by TTP. IL-10 mRNA levels were found to be elevated because of a reduced decay rate in primary macrophages from TTP-/- mice. Our study demonstrates the importance of experimental approaches for identifying targets of RNA-binding proteins.

NF-κB-Mediated MyoD Decay during Muscle Wasting Requires Nitric Oxide Synthase mRNA Stabilization, HuR Protein, and Nitric Oxide Release

ABSTRACT Muscle wasting (cachexia) is a consequence of chronic diseases, such as cancer, and is associated with degradation of muscle proteins such as MyoD. The cytokines tumor necrosis factor alpha and gamma interferon induce muscle degeneration by activating the transcription factor NF-κB and its target genes. Here, we show that a downstream target of NF-κB is the nitric oxide (NO) synthase gene (iNos) and suggest that NO production stimulates MyoD mRNA loss. In fact, although cytokine treatment of iNos−/− mice activated NF-κB, it did not trigger MyoD mRNA degeneration, demonstrating that NF-κB-mediated muscle wasting requires an active iNOS-NO pathway. The induced expression of iNOS by cytokines relies on both transcriptional activation via NF-κB and increased mRNA stability via the RNA-binding protein HuR. Moreover, we show that HuR regulates iNOS expression in an AMP-activated protein kinase (AMPK)-dependent manner. Furthermore, AMPK activation results in HuR nuclear sequestration, inhibition of iNOS synthesis, and reduction in cytokine-induced MyoD loss. These results define iNOS and HuR as critical players in cytokine-induced cachexia, establishing them as potential therapeutic targets.

Caspase-mediated cleavage of HuR in the cytoplasm contributes to pp32/PHAP-I regulation of apoptosis

The RNA-binding protein HuR affects cell fate by regulating the stability and/or the translation of messenger RNAs that encode cell stress response proteins. In this study, we delineate a novel regulatory mechanism by which HuR contributes to stress-induced cell death. Upon lethal stress, HuR translocates into the cytoplasm by a mechanism involving its association with the apoptosome activator pp32/PHAP-I. Depleting the expression of pp32/PHAP-I by RNA interference reduces both HuR cytoplasmic accumulation and the efficiency of caspase activation. In the cytoplasm, HuR undergoes caspase-mediated cleavage at aspartate 226. This cleavage activity is significantly reduced in the absence of pp32/PHAP-I. Substituting aspartate 226 with an alanine creates a noncleavable isoform of HuR that, when overexpressed, maintains its association with pp32/PHAP-I and delays the apoptotic response. Thus, we propose a model in which HuR association with pp32/PHAP-I and its caspase-mediated cleavage constitutes a regulatory step that contributes to an amplified apoptotic response.

An in-depth map of polyadenylation sites in cancer

We present a comprehensive map of over 1 million polyadenylation sites and quantify their usage in major cancers and tumor cell lines using direct RNA sequencing. We built the Expression and Polyadenylation Database to enable the visualization of the polyadenylation maps in various cancers and to facilitate the discovery of novel genes and gene isoforms that are potentially important to tumorigenesis. Analyses of polyadenylation sites indicate that a large fraction (∼30%) of mRNAs contain alternative polyadenylation sites in their 3′ untranslated regions, independent of the cell type. The shortest 3′ untranslated region isoforms are preferentially upregulated in cancer tissues, genome-wide. Candidate targets of alternative polyadenylation-mediated upregulation of short isoforms include POLR2K, and signaling cascades of cell–cell and cell–extracellular matrix contact, particularly involving regulators of Rho GTPases. Polyadenylation maps also helped to improve 3′ untranslated region annotations and identify candidate regulatory marks such as sequence motifs, H3K36Me3 and Pabpc1 that are isoform dependent and occur in a position-specific manner. In summary, these results highlight the need to go beyond monitoring only the cumulative transcript levels for a gene, to separately analysing the expression of its RNA isoforms.

A Boost for the Emerging Field of RNA Nanotechnology Report on the First International Conference on RNA Nanotechnology

This Nano Focus article highlights recent advances in RNA nanotechnology as presented at the First International Conference of RNA Nanotechnology and Therapeutics, which took place in Cleveland, OH, USA (October 23–25, 2010) (http://www.eng.uc.edu/nanomedicine/RNA2010/), chaired by Peixuan Guo and co-chaired by David Rueda and Scott Tenenbaum. The conference was the first of its kind to bring together more than 30 invited speakers in the frontier of RNA nanotechnology from France, Sweden, South Korea, China, and throughout the United States to discuss RNA nanotechnology and its applications. It provided a platform for researchers from academia, government, and the pharmaceutical industry to share existing knowledge, vision, technology, and challenges in the field and promoted collaborations among researchers interested in advancing this emerging scientific discipline. The meeting covered a range of topics, including biophysical and single-molecule approaches for characterization of RNA nanostructures; structure studies on RNA nanoparticles by chemical or biochemical approaches, computation, prediction, and modeling of RNA nanoparticle structures; methods for the assembly of RNA nanoparticles; chemistry for RNA synthesis, conjugation, and labeling; and application of RNA nanoparticles in therapeutics. A special invited talk on the well-established principles of DNA nanotechnology was arranged to provide models for RNA nanotechnology. An Administrator from National Institutes of Health (NIH) National Cancer Institute (NCI) Alliance for Nanotechnology in Cancer discussed the current nanocancer research directions and future funding opportunities at NCI. As indicated by the feedback received from the invited speakers and the meeting participants, this meeting was extremely successful, exciting, and informative, covering many groundbreaking findings, pioneering ideas, and novel discoveries.

Asymmetric Segregation of the Double-Stranded RNA Binding Protein Staufen2 during Mammalian Neural Stem Cell Divisions Promotes Lineage Progression

Asymmetric cell divisions are a fundamental feature of neural development, and misregulation can lead to brain abnormalities or tumor formation. During an asymmetric cell division, molecular determinants are segregated preferentially into one daughter cell to specify its fate. An important goal is to identify the asymmetric determinants in neural progenitor cells, which could be tumor suppressors or inducers of specific neural fates. Here, we show that the double-stranded RNA-binding protein Stau2 is distributed asymmetrically during progenitor divisions in the developing mouse cortex, preferentially segregating into the Tbr2(+) neuroblast daughter, taking with it a subset of RNAs. Knockdown of Stau2 stimulates differentiation and overexpression produces periventricular neuronal masses, demonstrating its functional importance for normal cortical development. We immunoprecipitated Stau2 to examine its cargo mRNAs, and found enrichment for known asymmetric and basal cell determinants, such as Trim32, and identified candidates, including a subset involved in primary cilium function.

Advances in RIP-Chip Analysis: RNA-Binding Protein Immunoprecipitation-Microarray Profiling

In eukaryotic organisms, gene regulatory networks require an additional level of coordination that links transcriptional and post-transcriptional processes. Messenger RNAs have traditionally been viewed as passive molecules in the pathway from transcription to translation. However, it is now clear that RNA-binding proteins (RBPs) play a major role in regulating multiple mRNAs to facilitate gene expression patterns. On this basis, post-transcriptional and transcriptional gene expression networks appear to be very analogous. Our previous research focused on targeting RBPs to develop a better understanding of post-transcriptional gene-expression processing and the regulation of mRNA networks. We developed technologies for purifying endogenously formed RBP-mRNA complexes from cellular extracts and identifying the associated messages using genome-scale, microarray technology, a method called ribonomics or RNA-binding protein immunoprecipitation-microarray (Chip) profiling or RIP-Chip. The use of the RIP-Chip methods has provided great insight into the infrastructure of coordinated eukaryotic post-transcriptional gene expression, insights which could not have been obtained using traditional RNA expression profiling approaches (1). This chapter describes the most current RIP-Chip techniques as we presently practice them. We also discuss some of the informatic aspects that are unique to analyzing RIP-Chip data.