Integrative regulatory mapping indicates that the RNA-binding protein HuR couples pre-mRNA processing and mRNA stability.

RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3' UTR binding sites were more highly stabilized than transcripts with only 3' UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in polypyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing.

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.

The Effect of Hepatocellular Carcinoma Surveillance in an Urban Population With Liver Cirrhosis.

BACKGROUND: Hepatocellular carcinoma (HCC) surveillance in patients with cirrhosis is aimed at early detection so that effective therapeutic options may be offered. We undertook this study to assess the patterns of surveillance that had been offered to HCC patients evaluated at our center, and the effect of these strategies on outcome. METHODS: Consecutive patients, age 18 years and older, diagnosed with HCC between December 2007 and December 2012 were identified. Surveillance was defined as α-fetoprotein measurement and/or imaging examination in the 12 months before HCC diagnosis. Logistic regression and survival analysis models were utilized to investigate the association of surveillance with patient characteristics and survival. RESULTS: A total of 305 patients with HCC and a background of cirrhosis were analyzed. HCC was detected by surveillance in 131 patients (43%). Of those who underwent surveillance, 92% were diagnosed with early-stage cancer (stages I and II) compared with 62% of those who did not undergo surveillance (P<0.001). The rate of surgical therapy (hepatic resection and liver transplantation) was almost doubled in the surveillance group (61% vs. 33%, P<0.05). At median follow-up of 27.3 months, overall survival was high at 55% and surveillance was significantly associated with longer survival (P=0.006). CONCLUSIONS: At our center, surveillance efficacy for HCC detection was notably higher than earlier reported. IMPACT: Patients who underwent surveillance were more likely to have their tumors detected at an early stage, to qualify for surgical therapy, and to have improved survival.

The viral and cellular microRNA targetome in lymphoblastoid cell lines.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, only a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis and/or lymphomagenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV strain B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3'UTRs. 531 of these sites contained seed matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3'UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBV-associated malignancies.

RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries.

Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3' and 5' ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1-249) and Rnl2(1-249)K227Q, for 3'-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5'-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3'-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5'-terminal 5-nt barcode extensions for a set of 20 barcoded 3' adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.

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

BACKGROUND: ZFP36, 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. RESULTS: Targets 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. CONCLUSIONS: ZFP36-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.

RNA targets of wild-type and mutant FET family proteins.

FUS, EWSR1 and TAF15, constituting the FET protein family, are abundant, highly conserved RNA-binding proteins with important roles in oncogenesis and neuronal disease, yet their RNA targets and recognition elements are unknown. Using PAR-CLIP, we defined global RNA targets for all human FET proteins and two ALS-causing human FUS mutants. FET members showed similar binding profiles, whereas FUS mutants showed a drastically altered binding pattern, consistent with changes in subcellular localization.

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

Primary effusion lymphoma (PEL) is caused by Kaposi's 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.