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Viruses and microRNAs: a toolbox for systematic analysis

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RNA silencing is emerging as a novel layer of regulation of virus–host interaction. Since individual small RNAs can probably repress dozens if not hundreds of target mRNA molecules, and transcripts, on the other hand, may be recognized by multiple regulatory small RNAs, a dense and complex interaction network of microRNAs (miRNAs) and their targets arises. A comprehensive analysis of miRNA functions thus not only requires systematic approaches employing high‐throughput technologies but also calls for the development of improved experimental technologies and a profound bioinformatic analysis. Integration of complementary approaches will enhance our understanding of the mutual regulation of virus and host. Focusing on herpesviruses, we here describe currently available technologies and summarize present results obtained by high‐throughput approaches. These techniques can be broadly applied to other virus families and pathways employing other classes of small regulatory RNAs and therefore are powerful universal tools for research on virus–host interaction. WIREs RNA 2011 2 787–801 DOI: 10.1002/wrna.92

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Figure 1.

The RNA silencing pathway and its manipulation by synthetic small regulatory RNAs. Cellular microRNAs (miRNAs) or miRNAs encoded by DNA viruses with nuclear replication typically originate in the nucleus where their hairpin precursors are produced from transcripts of specialized miRNA‐encoding genes or excised from introns. Following several cleavage steps executed by the RNases Drosha and Dicer and nuclear export mediated by Exportin‐5, an about 22‐nt long double‐stranded RNA intermediate is formed. The active strand of the miRNA (guide strand, red) is loaded onto the RNA‐induced silencing complex (RISC) and provides sequence specificity and mRNA binding properties of the effector complex. mRNAs with sufficient complementarity are recognized and bound by RISCs and suppressed. Depending on the degree of complementarity and other factors not depicted here, the message is degraded, destabilized, or translationally repressed. Small interfering RNAs (siRNAs) are usually excised in the cytoplasm from longer double‐stranded RNAs originating from endogenous or exogenous sources, but share large parts of their biogenesis and functional mechanisms with miRNAs. The RNA silencing pathway (gray arrows) can be manipulated by addition of synthetic double‐stranded miRNA mimics or siRNAs which functionally act like endogenous small RNAs. Alternatively, inhibitor molecules can be delivered which are recognized by the RISC instead of endogenous targets and lead to sustained RISC inhibition. Red arrows represent exogenous miRNA mimic and inhibitor molecules. The blue arrow represents exogenous siRNAs.

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Figure 2.

Methods applied for miRNA targetome analysis. Immunoprecipitation of RISC complexes followed by analysis of co‐purified RNA species identifies messages regulated by miRNAs (RIP‐Chip, black arrows). CLIP variants of the method employing UV cross‐linking, like HITS‐CLIP and PAR‐CLIP (blue arrows), specifically isolate RNA sequences within RISCs and can yield positional information of the actual miRNA binding site. CLIP, cross‐linking immunoprecipiation; HITS‐CLIP, high‐throughput sequencing of RNA isolated by cross‐linking immunoprecipitation; miRNA, microRNA; PAR‐CLIP, photoactivatable‐ribonucleoside‐enhanced cross‐linking and immunoprecipitation, RISC, RNA‐induced silencing complex.

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Figure 3.

Identification of microRNA (miRNA) targets using stable isotope‐labeling by amino acids in cell culture. Proteins are metabolically labeled in cell culture by replacing standard ‘light’ amino acids (green arrows) with amino acids of increased molecular weight (‘heavy’ amino acids, red arrows). Differently labeled proteins from two or more cell populations are distinguished and quantified in a comparative manner. These cell populations can be subjected to different treatments such as infection as shown here (yellow, virus particle). Alternatively, cell populations to be compared may display intrinsic differences such as ectopic gene expression of, for example, miRNAs or proteins relevant to viral infection. These external or intrinsic stimuli are expected to significantly alter protein expression levels in one cell population compared to the other which can be measured by mass spectrometry. By combining protein preparations immediately after cell lysis technical bias induced by subsequent sample processing is avoided. Since the weight difference of light and heavy amino acids is well defined, peptides originating from the two cell populations can be specifically identified and matched. Their relative amounts provide information on changes in protein abundance due to the treatment.

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