The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A/DDX2 RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of Silvestrol and related compounds. For example, eIF4A promotes T-ALL development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with Silvestrol has powerful therapeutic effects in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5′UTR sequences such as the 12-mer guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and Silvestrol-sensitive transcripts are a number of oncogenes, super-enhancer associated transcription factors, and epigenetic regulators. Hence, the 5′UTRs of selected cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

The DNA damage response can be initiated in response to a variety of stress signals that are encountered during physiological processes or in response to exogenous cues, such as ionizing radiation or DNA-damaging therapeutic agents. A number of methods have been developed to examine the morphological, biochemical, and molecular changes that take place during the DNA damage response. When cells are exposed to ionizing radiation or DNA-damaging chemotherapeutic agents, double-stranded breaks (DSBs) are generated that rapidly result in the phosphorylation of histone H2A variant H2AX. Because phosphorylation of H2AX at Ser 139 (γ-H2AX) is abundant, fast, and correlates well with each DSB, it is the most sensitive marker that can be used to examine the DNA damage produced and the subsequent repair of the DNA lesion. γ-H2AX can be detected by immunoblotting and immunostaining using microscopic or flow cytometric detection. Since γ-H2AX can be also generated during DNA replication, as a consequence of apoptosis, or as it is found associated with residual DNA damage, it is important to determine the kinetics, number, size, and morphology of γ-H2AX-associated foci. This chapter describes a few standard protocols that we have successfully used in our laboratory for a number of experimental systems, primarily hematologic and epithelial cells grown in culture.

SV, Dasarathy S. Hyperammonemia-mediated autophagy in skeletal muscle contributes to sarcopenia of cirrhosis. Am J Physiol Endocrinol Metab 303: E983-E993, 2012. First published August 14, 2012; doi:10.1152/ajpendo.00183.2012.-Hyperammonemia and sarcopenia (loss of skeletal muscle) are consistent abnormalities in cirrhosis and portosystemic shunting. We have shown that muscle ubiquitin-proteasome components are not increased with hyperammonemia despite sarcopenia. This suggests that an alternative mechanism of proteolysis contributes to sarcopenia in cirrhosis. We hypothesized that autophagy could be this alternative pathway since we observed increases in classic autophagy markers, increased LC3 lipidation, beclin-1 expression, and p62 degradation in immunoblots of skeletal muscle protein in cirrhotic patients. We observed similar changes in these autophagy markers in the portacaval anastamosis (PCA) rat model. To determine the mechanistic relationship between hyperammonemia and autophagy, we exposed murine C2C12 myotubes to ammonium acetate. Significant increases in LC3 lipidation, beclin-1 expression, and p62 degradation occurred by 1 h, whereas autophagy gene expression (LC3, Atg5, Atg7, beclin-1) increased at 24 h. C2C12 cells stably expressing GFP-LC3 or GFPmCherry-LC3 constructs showed increased formation of mature autophagosomes supported by electron microscopic studies. Hyperammonemia also increased autophagic flux in mice, as quantified by an in vivo autophagometer. Because hyperammonemia induces nitration of proteins in astrocytes, we quantified global muscle protein nitration in cirrhotic patients, in the PCA rat, and in C2C12 cells treated with ammonium acetate. Increased protein nitration was observed in all of these systems. Furthermore, colocalization of nitrated proteins with GFP-LC3-positive puncta in hyperammonemic C2C12 cells suggested that autophagy is involved in degradation of nitrated proteins. These observations show that increased skeletal muscle autophagy in cirrhosis is mediated by hyperammonemia and may contribute to sarcopenia of cirrhosis.

MotivationDeep sequencing based ribosome footprint profiling can provide novel insights into the regulatory mechanisms of protein translation. However, the observed ribosome profile is fundamentally confounded by transcriptional activity. In order to decipher principles of translation regulation, tools that can reliably detect changes in translation efficiency in case–control studies are needed.ResultsWe present a statistical framework and an analysis tool, RiboDiff, to detect genes with changes in translation efficiency across experimental treatments. RiboDiff uses generalized linear models to estimate the over-dispersion of RNA-Seq and ribosome profiling measurements separately, and performs a statistical test for differential translation efficiency using both mRNA abundance and ribosome occupancy.Availability and ImplementationRiboDiff webpage http://bioweb.me/ribodiff. Source code including scripts for preprocessing the FASTQ data are available at http://github.com/ratschlab/ribodiff.Supplementary information Supplementary data are available at Bioinformatics online.

IntroductionMetastasis is a complex disease involving a series of events that includes stimulation or repression of numerous gene products in a coordinate manner, resulting in the invasion of neoplastic cells and detachment from the primary tumors, penetration into blood and lymphatics, arrest by adhesion at distant sites, extravasation, induction of angiogenesis, evasion of host antitumor responses, and growth at metastatic sites [Nicolson, 1988]. To identify the genes potentially involved in breast cancer invasion and metastasis, Toh et al. performed differential screening of a cDNA library of metastatic and nonmetastatic adenocarcinoma cell lines from rat mammary glands and cloned a novel gene known as the metastasis-associated tumor gene (mta1) [Toh et al., 1994]. Despite widespread overexpression of the MTA1 protein in human tumors, the molecular functions of the MTA family remained a mystery until the proteomic analysis of the nucleosome remodeling and deacetylation (NuRD) complex identified MTA1 and MTA2 as integral to the histone deacetylase (HDAC) complexes [Xue et al., 1998;Zhang et al., 1998], thus providing clues about the role of the MTA family of proteins in chromatin remodeling. MTA3, the third member of the MTA family, was identified as an estrogen-inducible gene product that forms a distinct Mi-2/NuRD complex [Fujita et al., 2003]. MTA1s, a naturally occurring variant of MTA1, was accidentally discovered by researchers pursuing the role of MTA1 in transcriptional regulation of estrogen receptor-α (ER) [Kumar et al., 2002]. MTA1s is generated by alternative splicing at a cryptic splice-site in exon 14, involving the deletion of 47 base pair nucleotides, and a frame-shift involving the addition of 33 unique amino acids with no homology among sequences from Genbank. The resulting MTA1s gene product lacks the C-terminal region of other MTA family members [Kumar et al., 2002]. Using a polyclonal antibody against purified zymogen granule membrane components from rat pancreas, researchers identified ZG29p, a cDNA coding for the 29-kDa protein, by immunoscreening a hormonally stimulated pancreas cDNA library. ZG29p is an N-terminal truncated form of MTA1, coded by the last seven exons of MTA1 and present in the zymogen granules of the pancreas. ZG29p mediates an interaction with amylase and is involved in condensation-sorting in the exocrine rat pancreas [Kleene et al., 2000]. Domain structure of the MTA familyMTA1 and MTA2 are polypeptides with molecular masses of approximately 80 kDa and 70 kDa, respectively, whereas MTA3 is smaller, approximately 65 kDa. Protein alignment homology of the human MTA proteins revealed 68% and 73% homology of MTA2 and MTA3, respectively, with MTA1. The highest homology was concentrated in the N-terminal half of the proteins, whereas the C-terminal regions of the MTA proteins were divergent (Figure 1A and [Manavathi and Kumar, 2007] has not yet been directly tested; it is currently hypothesized from studies of proteins containing such domains. For example, the BAH domain ...