Daniel Beisang

Analysis of CUGBP1 Targets Identifies GU-Repeat Sequences That Mediate Rapid mRNA Decay

ABSTRACT CUG-repeat binding protein 1 (CUGBP1) mediates selective mRNA decay by binding to GU-rich elements (GREs) containing the sequence UGUUUGUUUGU found in the 3′ untranslated region (UTR) of short-lived transcripts. We used an anti-CUGBP1 antibody to immunoprecipitate CUGBP1 from HeLa cytoplasmic extracts and analyzed the associated transcripts using oligonucleotide microarrays. We identified 613 putative mRNA targets of CUGBP1 and found that the UGUUUGUUUGU GRE sequence and a GU-repeat sequence were both highly enriched in the 3′ UTRs of these targets. We showed that CUGBP1 bound specifically to the GU-repeat sequence and that insertion of this sequence into the 3′ UTR of a beta-globin reporter transcript conferred instability to the transcript. Based on these results, we redefined the GRE to include this GU-repeat sequence. Our results suggest that CUGBP1 coordinately regulates the mRNA decay of a network of transcripts involved in cell growth, cell motility, and apoptosis.

Regulation of CUG-binding Protein 1 (CUGBP1) Binding to Target Transcripts upon T Cell Activation

Background: We identified target transcripts of the RNA-binding protein CUGBP1 in resting and activated T cells. Results: T cell activation induced CUGBP1 phosphorylation, causing decreased CUGBP1 binding to target transcripts. Conclusion: CUGBP1 binding to a network of target transcripts is regulated by CUGBP1 phosphorylation following T cell activation. Significance: CUGBP1 target transcripts are coordinately regulated during T cell activation. The RNA-binding protein, CUG-binding protein 1 (CUGBP1), regulates gene expression at the levels of alternative splicing, mRNA degradation, and translation. We used RNA immunoprecipitation followed by microarray analysis to identify the cytoplasmic mRNA targets of CUGBP1 in resting and activated primary human T cells and found that CUGBP1 targets were highly enriched for the presence of GU-rich elements (GREs) in their 3′-untranslated regions. The number of CUGBP1 target transcripts decreased dramatically following T cell activation as a result of activation-dependent phosphorylation of CUGBP1 and decreased ability of CUGBP1 to bind to GRE-containing RNA. A large percentage of CUGBP1 target transcripts exhibited rapid and transient up-regulation, and a smaller percentage exhibited transient down-regulation following T cell activation. Many of the transiently up-regulated CUGBP1 target transcripts encode important regulatory proteins necessary for transition from a quiescent state to a state of cellular activation and proliferation. Overall, our results show that CUGBP1 binding to certain GRE-containing target transcripts decreased following T cell activation through activation-dependent phosphorylation of CUGBP1.

Perspectives on the ARE as it turns 25 years old

The AU‐rich element (ARE) was discovered in 1986 as a conserved mRNA sequence found in the 3′ untranslated region of the TNF‐α transcript and other transcripts encoding cytokines and inflammatory mediators. Shortly thereafter, the ARE was shown to function as a regulator of mRNA degradation, and AREs were later shown to regulate other posttranscriptional mechanisms such as translation and mRNA localization. AREs coordinately regulate networks of chemokine, cytokine, and growth regulatory transcripts involved in cellular activation, proliferation, and inflammation. ARE‐mediated regulation is carried out by a host of ARE‐binding proteins, whose activity is regulated in a cell type and activation‐dependent manner. The last 25 years of ARE research has offered insight into the mechanisms and regulation of ARE‐mediated mRNA decay, and has provided a road map for the discovery of additional mRNA regulatory motifs. The future of ARE research will transition from a discovery phase to a phase focused on translating basic biological findings into novel therapeutic targets. Our understanding of ARE‐mediated gene regulation and posttranscriptional control has implications for many fields of study including developmental biology, neuroscience, immunobiology, and cancer biology. WIREs RNA 2012 doi: 10.1002/wrna.1125

Global assessment of GU-rich regulatory content and function in the human transcriptome.

Unlike AU-rich elements (AREs) that are largely present in the 3’UTRs of many unstable mammalian mRNAs, the function and abundance of GU-rich elements (GREs) are poorly understood. We performed a genome-wide analysis and found that at least 5% of human genes contain GREs in their 3’UTRs with functional over-representation in genes involved in transcription, nucleic acid metabolism, developmental processes, and neurogenesis. GREs have similar sequence clustering patterns with AREs such as overlapping GUUUG pentamers and enrichment in 3′UTRs. Functional analysis using T-cell mRNA expression microarray data confirms correlation with mRNA destabilization. Reporter assays show that compared to AREs the ability of GREs to destabilize mRNA is modest and does not increase with the increasing number of overlapping pentamers. Naturally occurring GREs within U-rich contexts were more potent in destabilizing GFP reporter mRNAs than synthetic GREs with perfectly overlapping pentamers. Overall, we find that GREs bear a resemblance to AREs in sequence patterns but they regulate a different repertoire of genes and have different dynamics of mRNA decay. A dedicated resource on all GRE-containing genes of the human, mouse and rat genomes can be found at brp.kfshrc.edu.sa/GredOrg.

CELF1, a Multifunctional Regulator of Posttranscriptional Networks

In order to assure the precise utilization of genetic information, gene expression is regulated at the level of transcription as well as multiple post-transcriptional levels including splicing, transport, localization, mRNA stability, and translation [1],[2],[3],[4],[5],[6],[7]. During evolution, cells developed precise mechanisms to ensure that each transcript is appropriately stored, modified, translated or degraded, depending on the need for the mRNA or encoded protein by the cell. Steady-state protein levels within a cell correlate poorly with steady-state levels of mRNA, leading scientists to hypothesize that the gene expression is regulated at post-transcriptional levels [8]. Work over the past quarter century has resulted in the identification of unifying concepts in post-transcriptional regulation. One unifying concept states is that post-transcriptional regulation is mediated by two major molecular components: cis-acting regulatory sequence elements and trans-acting factors. Cisacting regulatory sequence elements are sub-sequences contained in the 5’ untranslated region (UTR), coding region, and 3’UTR of mRNA that are selectively recognized by a complementary set of one or more trans-acting factors to regulate post-transcriptional gene expression. Trans-acting factors include RNA-binding proteins (RBPs) and microRNAs (miRNAs) which are able to influence the fate of mRNA by controlling processes such as translation and mRNA degradation (reviewed in references [9],[10],[11],[12]). The combinatorial interplay between various miRNAs and RBPs binding to a given mRNA allows for the transcript specific regulation critical to many cellular decisions during development [13],[14],[15],[16] and in response to environmental stimuli (reviewed in references [17],[18],[19],[20],[21],[22]).

Mesenchymal stem cells in the pathogenesis and treatment of bronchopulmonary dysplasia: A clinical review

Advances in neonatal medicine have led to increased survival of infants born at the limits of viability, resulting in an increased incidence of bronchopulmonary dysplasia (BPD). BPD is a chronic lung disease of premature infants characterized by the arrest of alveolarization, fibroblast activation, and inflammation. BPD leads to significant morbidity and mortality in the neonatal period and is one of the leading causes of chronic lung disease in children. The past decade has brought a surge of trials investigating cellular therapies for the treatment of pulmonary diseases. Mesenchymal stem cells (MSCs) are of particular interest because of their ease of isolation, low immunogenicity, and anti-inflammatory and reparative properties. Clinical trials of MSCs have demonstrated short-term safety and tolerability; however, studies have also shown populations of MSCs with adverse pro-inflammatory and myofibroblastic characteristics. Cell-based therapies may represent the next breakthrough therapy for the treatment of BPD, however, there remain barriers to implementation as well as gaps in knowledge of the role of endogenous MSCs in the pathogenesis of BPD. Concurrent high-quality basic science, translational, and clinical studies investigating the fundamental pathophysiology underlying BPD, therapeutic mechanisms of exogenous MSCs, and logistics of translating cellular therapies will be important areas of future research.

The hepatitis C viral nonstructural protein 5A stabilizes growth-regulatory human transcripts

Abstract Numerous mammalian proto-oncogene and other growth-regulatory transcripts are upregulated in malignancy due to abnormal mRNA stabilization. In hepatoma cells expressing a hepatitis C virus (HCV) subgenomic replicon, we found that the viral nonstructural protein 5A (NS5A), a protein known to bind to viral RNA, also bound specifically to human cellular transcripts that encode regulators of cell growth and apoptosis, and this binding correlated with transcript stabilization. An important subset of human NS5A-target transcripts contained GU-rich elements, sequences known to destabilize mRNA. We found that NS5A bound to GU-rich elements in vitro and in cells. Mutation of the NS5A zinc finger abrogated its GU-rich element-binding and mRNA stabilizing activities. Overall, we identified a molecular mechanism whereby HCV manipulates host gene expression by stabilizing host transcripts in a manner that would promote growth and prevent death of virus-infected cells, allowing the virus to establish chronic infection and lead to the development of hepatocellular carcinoma.

Bacterial growth and antibiotic resistance patterns in cirrhotic ascites

of time). Of these samples, 28 were from subjects that had ascites secondary to liver cirrhosis, and 24 were from individuals with postoperative complications or peritoneal dialysis. Of the 28 samples from cirrhotic patients, which are further described below, 24 had a serum albumin-toascites gradient (SAAG) compatible with ascites from portal hypertension. All patients were males with a median age of 59 years (IQR 53–62) and median SAAG was 1.7 (IQR 1.3–2.2). The cause of cirrhosis was unknown in 12 cases (40%) and the most common known cause of cirrhosis was alcohol abuse (40%), followed by HCV (14%) and hemochromatosis (7%). Seven of the twenty-eight samples (25%) had >250 PMNs/mm compatible with SBP (median 400 cells/mm). None of the patients had fever on presentation, and only seven patients (25%) had abdominal pain. The presence of abdominal pain was not related to PMN content in ascitic fluid. Of 28 samples with positive growth in cirrhotic patients, 5 grew more than one organism. The most commonly identified organisms were coagulase negative Staphylococci (N:12) and Viridans streptococci (N:6), followed by Escherichia coli (N:4) and other Gram-negative organisms (Propionibacterium acnes, 4; Klebsiella sp, 2; Diphtheroids, S. anginosus, Acinetobacter, Stenotrophomonas, Veillonella, Bacteroides dorei, Prevotella bucae, Enterococcus Faecium, 1 each). Almost 40% of our samples grew coagulase negative Staphylococcus. We could not rule out that some of this growth is related to sample contamination. However, a bacterial flora of predominant Gram-positive cocci in both SBP and bacterascites was recently reported in a prospective observational French study by Piroth et al. in alignment with our findings [4]. Overall, our samples showed a lower number of Gram-negative bacteria than generally reported for SBP [4, 5]. This finding could be related to extraction technique, the veteran population, or difference in antibiotic use in the Veterans To the Editor,