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Abstract: Small RNAs (21–24 nucleotides) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) are potent regulators of gene expression in both plants and animals. Several hundred genes encoding miRNAs and thousands of siRNAs have been experimentally identified by cloning approaches. New sequencing technologies facilitate the identification of these molecules and provide global quantitative expression data in a given biological sample. Here, we describe the methods used in our laboratory to construct small RNA cDNA libraries for high-throughput sequencing using technologies such as MPSS, 454 or SBS. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) are ∼21nt small RNAs that pair to their target mRNAs and in many cases trigger cleavage, particularly in plants. Although many computational tools can predict miRNA:mRNA interactions, it remains critical to validate cleavage events, due to miRNA function in translational repression or due to high rates of false positives (over 90%) for unvalidated target predictions. A few years ago, three laboratories described similar methods to validate cleavage of miRNA targets by the cloning en masse of 5′ ends of cleaved or uncapped mRNAs. To take advantage of the recent progress in high-throughput sequencing technology, we have devised an updated protocol to (1) enable much faster library preparation, and (2) reduce the cost by pooling indexed samples together for sequencing. Here we provide a step-by-step protocol for PARE library construction, starting from total RNA. This protocol has been successfully used in our laboratory to validate miRNA targets in a variety of plant species. We also provide advice for troubleshooting on some common issues. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) are small RNAs that derive from endogenous precursors harboring foldback structures. Plant miRNA precursors are quite variable in their size and shape. Still, the miRNA processing machinery, consisting of DICER-LIKE1 (DCL1) and accessory proteins recognize structural features on the precursors to cleave them at specific places releasing the mature miRNAs. The identification of miRNA processing intermediates in plants has mostly relied on a modified 5′ RACE method, designed to detect the 5′ end of uncapped RNAs. However, this method is time consuming and is, therefore, only practical for the analysis of a handful miRNAs. Here, we present a modification of this approach in order to perform genome-wide analysis of miRNA processing intermediates. Briefly, a reverse transcription is performed with a mixture of specific primers designed against all known miRNA precursors. miRNA processing intermediates are then specifically amplified to generate a library and subjected to deep sequencing. This method, called SPARE (Specific Parallel Amplification of 5′ RNA Ends) allows the identification of processing intermediates for most of the Arabidopsis miRNAs. The results enable the determination of the DCL1 processing direction and the cleavage sites introduced by miRNA processing machinery in the precursors. The SPARE method can be easily adapted to detect miRNA-processing intermediates in other systems. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) are small RNAs that derive from endogenous precursors harboring foldback structures. Plant miRNA precursors are quite variable in their size and shape. Still, the miRNA processing machinery, consisting of DICER-LIKE1 (DCL1) and accessory proteins recognize structural features on the precursors to cleave them at specific places releasing the mature miRNAs. The identification of miRNA processing intermediates in plants has mostly relied on a modified 5′ RACE method, designed to detect the 5′ end of uncapped RNAs. However, this method is time consuming and is, therefore, only practical for the analysis of a handful miRNAs. Here, we present a modification of this approach in order to perform genome-wide analysis of miRNA processing intermediates. Briefly, a reverse transcription is performed with a mixture of specific primers designed against all known miRNA precursors. miRNA processing intermediates are then specifically amplified to generate a library and subjected to deep sequencing. This method, called SPARE (Specific Parallel Amplification of 5′ RNA Ends) allows the identification of processing intermediates for most of the Arabidopsis miRNAs. The results enable the determination of the DCL1 processing direction and the cleavage sites introduced by miRNA processing machinery in the precursors. The SPARE method can be easily adapted to detect miRNA-processing intermediates in other systems. [Copyright &y& Elsevier]

Abstract: Viral microRNAs regulate gene expression using either translational repression or mRNA cleavage and decay. Two microRNAs from infectious laryngotracheitis virus (ILTV), iltv-miR-I5 and iltv-miR-I6, map antisense to the ICP4 gene. Post-transcriptional repression by these microRNAs was tested against a portion of the ICP4 coding sequence cloned downstream of firefly luciferase. Luciferase activity was downregulated by approximately 60% with the iltv-miR-I5 mimic. Addition of an iltv-miR-I5 antagomiR or mutagenesis of the target seed sequence alleviated this effect. The iltv-miR-I5 mimic, when co-transfected with a plasmid expressing ICP4, reduced ICP4 transcript levels by approximately 50%, and inhibition was relieved by an iltv-miR-I5 antagomiR. In infected cells, iltv-miR-I5 mediated cleavage at the canonical site, as indicated by modified RACE analysis. Thus, in this system, iltv-miR-I5 decreased ILTV ICP4 mRNA levels via transcript cleavage and degradation. Downregulation of ICP4 could impact the balance between the lytic and latent states of the virus in vivo. [Copyright &y& Elsevier]

Abstract: Many herpesviruses, including Marek''s disease viruses (MDV1 and MDV2), encode microRNAs. In this study, we report microRNAs of two related herpesviruses, infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT), as well as additional MDV2 microRNAs. The genome locations, but not microRNA sequences, are conserved among all four of these avian herpesviruses. Most are clustered in the repeats flanking the unique long region (I/TRL), except in ILTV which lacks these repeats. Two abundant ILTV microRNAs are antisense to the immediate early gene ICP4. A homologue of host microRNA, gga-miR-221, was found among the HVT microRNAs. Additionally, a cluster of HVT microRNAs was found in a region containing two locally duplicated segments, resulting in paralogous HVT microRNAs with 96–100% identity. The prevalence of microRNAs in the genomic repeat regions as well as in local repeats suggests the importance of genetic plasticity in herpesviruses for microRNA evolution and preservation of function. [Copyright &y& Elsevier]