Neelanjan Mukherjee

A viral microRNA functions as an orthologue of cellular miR-155

All metazoan eukaryotes express microRNAs (miRNAs), roughly 22-nucleotide regulatory RNAs that can repress the expression of messenger RNAs bearing complementary sequences. Several DNA viruses also express miRNAs in infected cells, suggesting a role in viral replication and pathogenesis. Although specific viral miRNAs have been shown to autoregulate viral mRNAs or downregulate cellular mRNAs, the function of most viral miRNAs remains unknown. Here we report that the miR-K12-11 miRNA encoded by Kaposi’s-sarcoma-associated herpes virus (KSHV) shows significant homology to cellular miR-155, including the entire miRNA ‘seed’ region. Using a range of assays, we show that expression of physiological levels of miR-K12-11 or miR-155 results in the downregulation of an extensive set of common mRNA targets, including genes with known roles in cell growth regulation. Our findings indicate that viral miR-K12-11 functions as an orthologue of cellular miR-155 and probably evolved to exploit a pre-existing gene regulatory pathway in B cells. Moreover, the known aetiological role of miR-155 in B-cell transformation suggests that miR-K12-11 may contribute to the induction of KSHV-positive B-cell tumours in infected patients.

FMRP targets distinct mRNA sequence elements to regulate protein expression

Fragile X syndrome (FXS) is a multi-organ disease that leads to mental retardation, macro-orchidism in males and premature ovarian insufficiency in female carriers. FXS is also a prominent monogenic disease associated with autism spectrum disorders (ASDs). FXS is typically caused by the loss of fragile X mental retardation 1 (FMR1) expression, which codes for the RNA-binding protein FMRP. Here we report the discovery of distinct RNA-recognition elements that correspond to the two independent RNA-binding domains of FMRP, in addition to the binding sites within the messenger RNA targets for wild-type and I304N mutant FMRP isoforms and the FMRP paralogues FXR1P and FXR2P (also known as FXR1 and FXR2). RNA-recognition-element frequency, ratio and distribution determine target mRNA association with FMRP. Among highly enriched targets, we identify many genes involved in ASD and show that FMRP affects their protein levels in human cell culture, mouse ovaries and human brain. Notably, we discovered that these targets are also dysregulated in Fmr1−/− mouse ovaries showing signs of premature follicular overdevelopment. These results indicate that FMRP targets share signalling pathways across different cellular contexts. As the importance of signalling pathways in both FXS and ASD is becoming increasingly apparent, our results provide a ranked list of genes as basis for the pursuit of new therapeutic targets for these neurological disorders.

Viral microRNA targetome of KSHV-infected primary effusion lymphoma cell lines.

Primary effusion lymphoma (PEL) is caused by Kaposis sarcoma-associated herpesvirus (KSHV) and frequently also harbors Epstein-Barr virus (EBV). The expression of KSHV- and EBV-encoded microRNAs (miRNAs) in PELs suggests a role for these miRNAs in latency and lymphomagenesis. Using PAR-CLIP, a technology which allows the direct and transcriptome-wide identification of miRNA targets, we delineate the target sites for all viral and cellular miRNAs expressed in PEL cell lines. The resulting data set revealed that KSHV miRNAs directly target more than 2000 cellular mRNAs, including many involved in pathways relevant to KSHV pathogenesis. Moreover, 58% of these mRNAs are also targeted by EBV miRNAs, via distinct binding sites. In addition to a known viral analog of cellular miR-155, we show that KSHV encodes a viral miRNA that mimics cellular miR-142-3p function. In summary, this study identifies an extensive list of KSHV miRNA targets, which are likely to influence viral replication and pathogenesis.

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/.

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

ABSTRACT 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.

Systematic analysis of posttranscriptional gene expression

Recent systems studies of gene expression have begun to dissect the layers of regulation that underlie the eukaryotic transcriptome, the combined consequence of transcriptional and posttranscriptional events. Among the regulatory layers of the transcriptome are those of the ribonome, a highly dynamic environment of ribonucleoproteins in which RNA‐binding proteins (RBPs), noncoding regulatory RNAs (ncRNAs) and messenger RNAs (mRNAs) interact. While multiple mRNAs are coordinated together in groups within the ribonome of a eukaryotic cell, each individual type of mRNA consists of multiple copies, each of which has an opportunity to be a member of more than one modular group termed a posttranscriptional RNA operon or regulon (PTRO). The mRNAs associated with each PTRO encode functionally related proteins and are coordinated at the levels of RNA stability and translation by the actions of the specific RBPs and noncoding regulatory RNAs. This article examines the methods that led to the elucidation of PTROs and the coordinating mechanisms that appear to regulate the RNA components of PTROs. Moreover, the article considers the characteristics of the dynamic systems that drive PTROs and how mRNA components are bound collectively in physical ‘states’ to respond to cellular perturbations and diseases. In conclusion, these studies have challenged the extent to which cellular mRNA abundance can inform investigators of the functional status of a biological system. We argue that understanding the ribonome has greater potential for illuminating the underlying coordination principles of growth, differentiation, and disease. Copyright

Detecting actively translated open reading frames in ribosome profiling data

RNA-sequencing protocols can quantify gene expression regulation from transcription to protein synthesis. Ribosome profiling (Ribo-seq) maps the positions of translating ribosomes over the entire transcriptome. We have developed RiboTaper (available at https://ohlerlab.mdc-berlin.de/software/), a rigorous statistical approach that identifies translated regions on the basis of the characteristic three-nucleotide periodicity of Ribo-seq data. We used RiboTaper with deep Ribo-seq data from HEK293 cells to derive an extensive map of translation that covered open reading frame (ORF) annotations for more than 11,000 protein-coding genes. We also found distinct ribosomal signatures for several hundred upstream ORFs and ORFs in annotated noncoding genes (ncORFs). Mass spectrometry data confirmed that RiboTaper achieved excellent coverage of the cellular proteome. Although dozens of novel peptide products were validated in this manner, few of the currently annotated long noncoding RNAs appeared to encode stable polypeptides. RiboTaper is a powerful method for comprehensive de novo identification of actively used ORFs from Ribo-seq data.

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

SummaryGlioblastoma 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.

Coordinated posttranscriptional mRNA population dynamics during T-cell activation

Although RNA‐binding proteins (RBPs) coordinate many key decisions during cell growth and differentiation, the dynamics of RNA–RBP interactions have not been extensively studied on a global basis. We immunoprecipitated endogenous ribonucleoprotein complexes containing HuR and PABP throughout a T‐cell activation time course and identified the associated mRNA populations using microarrays. We used Gaussian mixture modeling as a discriminative model, treating RBP association as a discrete variable (target or not target), and as a generative model, treating RBP‐association as a continuous variable (probability of association). We report that HuR interacts with different populations of mRNAs during T‐cell activation. These populations encode functionally related proteins that are members of the Wnt pathway and proteins mediating T‐cell receptor signaling pathways. Moreover, the mRNA targets of HuR were found to overlap with the targets of other posttranscriptional regulatory factors, indicating combinatorial interdependence of posttranscriptional regulatory networks and modules after activation. Applying HuR mRNA dynamics as a quantitative phenotype in the drug‐gene‐phenotype Connectivity Map, we identified candidate small molecule effectors of HuR and T‐cell activation. We show that one of these candidates, resveratrol, exerts T‐cell activation‐dependent posttranscriptional effects that are rescued by HuR. Thus, we describe a strategy to systematically link an RBP and condition‐specific posttranscriptional effects to small molecule drugs.

Global target mRNA specification and regulation by the RNA-binding protein ZFP36.

BackgroundZFP36, also known as tristetraprolin or TTP, and ELAVL1, also known as HuR, are two disease-relevant RNA-binding proteins (RBPs) that both interact with AU-rich sequences but have antagonistic roles. While ELAVL1 binding has been profiled in several studies, the precise in vivo binding specificity of ZFP36 has not been investigated on a global scale. We determined ZFP36 binding preferences using cross-linking and immunoprecipitation in human embryonic kidney cells, and examined the combinatorial regulation of AU-rich elements by ZFP36 and ELAVL1.ResultsTargets bound and negatively regulated by ZFP36 include transcripts encoding proteins necessary for immune function and cancer, and transcripts encoding other RBPs. Using partial correlation analysis, we were able to quantify the association between ZFP36 binding sites and differential target RNA abundance upon ZFP36 overexpression independent of effects from confounding features. Genes with increased mRNA half-lives in ZFP36 knockout versus wild-type mouse cells were significantly enriched for our human ZFP36 targets. We identified thousands of overlapping ZFP36 and ELAVL1 binding sites, in 1,313 genes, and found that ZFP36 degrades transcripts through specific AU-rich sequences, representing a subset of the U-rich sequences ELAVL1 interacts with to stabilize transcripts.ConclusionsZFP36-RNA target specificities in vivo are quantitatively similar to previously reported in vitro binding affinities. ZFP36 and ELAVL1 bind an overlapping spectrum of RNA sequences, yet with differential relative preferences that dictate combinatorial regulatory potential. Our findings and methodology delineate an approach to unravel in vivo combinatorial regulation by RNA-binding proteins.