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In deciphering the regulatory networks of gene expression controlled by the small non-coding RNAs known as microRNAs (miRNAs), a major challenge has been with the identification of the true mRNA targets by these RNAs within the context of the enormous numbers of predicted targets for each of these small RNAs. To facilitate the system-wide identification of miRNA targets, a variety of system wide methods, such as proteomics, have been implemented. Here we describe the utilization of quantitative label-free proteomics and bioinformatics to identify the most significant changes to the proteome upon expression of the miR-23a-27a-24-2 miRNA cluster. In light of recent work leading to the hypothesis that only the most pronounced regulatory events by miRNAs may be physiologically relevant, our data reveal that label-free analysis circumvents the limitations of proteomic labeling techniques that limit the maximum differences that can be quantified. The result of our analysis identifies a series of novel candidate targets that are reduced in abundance by more than an order of magnitude upon the expression of the miR-23a-27a-24-2 cluster.

MicroiRNAs are genome encoded small double stranded RNAs that regulate expression of homologous mRNAs. With approximately 2500 human miRNAs and each having hundreds of potential mRNA targets, miRNA based gene regulation is quite pervasive in both development and disease. While there are numerous studies investigating miRNA:mRNA and miRNA:protein target expression correlations, there are relatively few studies of miRNA:miRNA co-expression. Here we report on our analysis of miRNA:miRNA co-expression using expression data from the miRNA expression atlas of Landgraf et al. Our analysis indicates that many, but not all, genomically clustered miRNAs are co-expressed as a single pri-miRNA transcript. We have also identified co-expression groups that have similar biological activity. Further, the non-correlative miRNAs we have uncovered have been shown to be of utility in establishing miRNA biomarkers and signatures for certain tumours and cancers.

Argonaute 2 (Ago2) protein is a central effector of RNA interference (RNAi) pathways and regulates mammalian genes on a global level. The mechanisms of Ago2-mediated silencing are well understood, but less is known about its regulation. Recent reports indicate that phosphorylation significantly affects Ago2 activity. Here, we investigated the effect of mutating all known phospho-residues within Ago2 on its localization and activity. Ago2 associates with two different cytoplasmic RNA granules known as processing bodies (P-bodies) and stress granules, but the nature of this phenomenon is controversial. We report that replacing serine with a phospho-mimetic aspartic acid at position 798 completely abrogates association of Ago2 with P-bodies and stress granules. The effect of this mutation on its activity in gene silencing was modest, which was surprising because association of Ago2 with cytoplasmic RNA granules is thought to be a consequence of its role in RNAi. As such, our data indicate that targeting of Ago2 to P-bodies and stress granules is separable from its role in RNAi and likely requires dynamic phosphorylation of serine 798., (© 2015 Lopez-Orozco et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Antisense RNAs have long been known to regulate diverse aspects of plasmid biology. Here we review the FinOP system that modulates F plasmid gene expression through regulation of the F plasmid transcription factor, TraJ. FinOP is a two component system composed of an antisense RNA, FinP, which represses TraJ translation, and a protein, FinO, which is required to stabilize FinP and facilitate its interactions with its traJ mRNA target. We review the evidence that FinO acts as an RNA chaperone to bind and destabilize internal stem-loop structures within the individual RNAs that would otherwise block intermolecular RNA duplexing. Recent structural studies have provided mechanistic insights into how FinO may facilitate interactions between FinP and traJ mRNA. We also review recent findings that two other proteins, Escherichia coli ProQ and Neisseria meningitidis NMB1681, may represent FinO-like RNA chaperones., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Rationale: Tandem mass (MS/MS) spectra generated by collision-induced dissociation (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues., Methods: Here we introduce arginyl-tRNA protein transferase (ATE, EC 2.3.2.8) for proteomics. ATE recognizes acidic amino acids or oxidized Cys at the N-terminus of a substrate peptide and conjugates an arginine from an aminoacylated tRNA(Arg) onto the N-terminus of the substrate peptide. This enzymatic reaction is carried out under physiological conditions and, in combination with Lys-C/Asp-N double digest, results in arginylated peptides with basic amino acids on both termini., Results: We demonstrate that in vitro arginylation of peptides using yeast arginyl tRNA protein transferase 1 (yATE1) is a robust enzymatic reaction, specific to only modifying N-terminal acidic amino acids. Precursors originating from arginylated peptides generally have an increased protonation state compared with their non-arginylated forms. Furthermore, the product ion spectra of arginylated peptides show near complete 2× fragment ladders within the same MS/MS spectrum using commonly available electrospray ionization peptide fragmentation modes. Unexpectedly, arginylated peptides generate complete y- and c-ion series using electron transfer dissociation (ETD) despite having an internal proline residue., Conclusions: We introduce a rapid enzymatic method to generate peptides flanked on either terminus by basic amino acids, resulting in a rich, redundant MS/MS fragment pattern., (© 2014 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

While functional mature microRNAs (miRNAs) are small ∼22 base oligonucleotides that target specific mRNAs, miRNAs are initially expressed as long transcripts (pri-miRNAs) that undergo sequential processing to yield the mature miRNAs. We have previously reported that the pri-miR-17∼92 cluster adopts a compact globular folded structure that internalizes a 3' core domain resulting in reduced miRNA maturation and subsequent mRNA targeting. Using a site-specific photo-cross-linker we have identified a tertiary contact within the 3' core domain of the pri-miRNA between a non-miRNA stem-loop and the pre-miR-19b hairpin. This tertiary contact is involved in the formation of the compact globular fold of the cluster while its disruption enhances miR-92a expression and mRNA targeting. We propose that this tertiary contact serves as a molecular scaffold to restrict expression of the proposed antiangiogenic miR-92a, allowing for the overall pro-angiogenic effect of miR-17∼92 expression.

MicroRNAs (miRNAs) are genome-encoded small double-stranded RNAs that have emerged as key regulators of gene expression and are implicated in most aspects of human development and disease. Canonical miRNA biogenesis involves processing of ∼70-nucleotide pre-miRNA hairpins by Dicer to generate mature ∼22-nucleotide miRNAs, which target complementary RNA sequences. Despite the importance of miRNA biogenesis, signaling mechanisms controlling this process are poorly defined. Here we demonstrate that the post-transcriptional regulation of Dicer is controlled by the cell density-mediated localization of the Hippo pathway effectors TAZ (transcriptional co-activator with PDZ-binding motif) and YAP (Yes-associated protein) (TAZ/YAP). We show that nuclear TAZ/YAP, which are abundant at low cell density, are required for efficient pre-miRNA processing. Knockdown of TAZ/YAP in low density cells, or density-mediated sequestration of TAZ/YAP into the cytoplasm, results in the defective processing of pre-miRNAs. Strikingly, one exception is Let-7, which accumulates upon loss of nuclear TAZ/YAP, leading to Let-7-dependent reduction in Dicer levels. Accordingly, inhibition of Let-7 rescues the miRNA biogenesis defects observed following TAZ/YAP knockdown. Thus, density-regulated TAZ/YAP localization defines a critical and previously unrecognized mechanism by which cells relay cell contact-induced cues to control miRNA biogenesis.

The understanding of RNA in regulating gene expression has exploded over the past 15 years. MicroRNAs (miRNAs) have vastly expanded the role of RNA in gene regulation beyond spliceosomal, ribosomal, and messenger RNAs. Approximately one half of miRNAs are polycistronic, where two or more miRNAs are encoded on a single pri-miRNA transcript, termed a miRNA cluster. The six miRNAs of the miR-17~92 cluster are contained within a ~800 nucleotide region within intron 3 of the cl13orf25 ~7 kb pri-miRNA transcript. We recently reported on the tertiary structured domain of miR-17~92 and its role in modulating miRNA biogenesis. The key finding was that the cluster structure explained the differential processing of the miRNA hairpins by Drosha. This work demonstrated the need to consider pri-miRNA tertiary structure in miRNA biogenesis. Since biochemical structure probing is typically performed on relatively short RNAs (≤200 nucleotides), we had to adapt these methodologies for application on large RNAs (~800 nucleotide miR-17~92 pri-miRNA). We present here our adaptation of a protection footprinting method using ribonucleases to probe the structure of the ~800 nucleotide miR-17~92 pri-miRNA. We outline the technical difficulties involved in probing large RNAs and data visualization using denaturing polyacrylamide gel electrophoresis and how we adapted the existing approaches to probe large RNAs. The methodology outlined here is generally applicable to large RNAs including long noncoding RNAs (lncRNA).

MicroRNAs (miRNAs) regulate gene expression in a variety of biological pathways such as development and tumourigenesis. miRNAs are initially expressed as long primary transcripts (pri-miRNAs) that undergo sequential processing by Drosha and then Dicer to yield mature miRNAs. miR-17~92 is a miRNA cluster that encodes 6 miRNAs and while it is essential for development it also has reported oncogenic activity. To date, the role of RNA structure in miRNA biogenesis has only been considered in terms of the secondary structural elements required for processing of pri-miRNAs by Drosha. Here we report that the miR-17~92 cluster has a compact globular tertiary structure where miRNAs internalized within the core of the folded structure are processed less efficiently than miRNAs on the surface of the structure. Increased miR-92 expression resulting from disruption of the compact miR-17~92 structure results in increased repression of integrin α5 mRNA, a known target of miR-92a. In summary, we describe the first example of pri-miRNA structure modulating differential expression of constituent miRNAs.

Transporter ProP mediates osmolyte accumulation in Escherichia coli cells exposed to high osmolality media. The cytoplasmic ProQ protein amplifies ProP activity by an unknown mechanism. The N- and C-terminal domains of ProQ are predicted to be structurally similar to known RNA chaperone proteins FinO and Hfq from E. coli. Here we demonstrate that ProQ is an RNA chaperone, binding RNA and facilitating both RNA strand exchange and RNA duplexing. Experiments performed with the isolated ProQ domains showed that the FinO-like domain serves as a high-affinity RNA-binding domain, whereas the Hfq-like domain is largely responsible for RNA strand exchange and duplexing. These data suggest that ProQ may regulate ProP production. Transcription of proP proceeds from RpoD- and RpoS-dependent promoters. Lesions at proQ affected ProP levels in an osmolality- and growth phase-dependent manner, decreasing ProP levels when proP was expressed from its own chromosomal promoters or from a heterologous plasmid-based promoter. Small RNA molecules are known to regulate cellular levels of sigma factor RpoS. ProQ did not act by changing RpoS levels since proQ lesions did not influence RpoS-dependent stationary phase thermotolerance and they affected ProP production and activity similarly in bacteria without and with an rpoS defect. Taken together, these results suggest that ProQ does not regulate proP transcription. It may act as an RNA-binding protein to regulate proP translation.

This protocol describes a general method for the preparation of RNAs in which the reactivity or hydrogen-bonding properties of the molecule are modified in a photoreversible fashion by use of a caging strategy. A single caged adenosine, modified at the 2' position as a nitro-benzyl ether, can be incorporated into short RNAs by chemical synthesis or into long RNAs by a combination of chemical and enzymatic synthesis. The modified RNAs can be uncaged by photolysis under a variety of conditions including the use of a laser or xenon lamp, and the course of this uncaging reaction may be readily followed by HPLC or thin-layer chromatography.

Getting spliced? Pre-mRNA splicing is catalyzed by the spliceosome, a complex assembly of proteins and RNA, which forms in an ordered fashion on the substrate. If one of the residues of the substrate is caged with a photolabile o-nitrobenzyl group, the splicing reaction can be transiently blocked, until subsequent initiation by photolysis of the complexes. This RNA-caging approach effectively separates the spliceosome assembly from the catalytic reaction and allows the two processes to be studied independently., (© 2001 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.)

The biological activity of large RNAs is dependent on the formation of complex folded structures that determine function. Typically the creation of such structures requires divalent magnesium and in many cases the folding process takes place over the course of several minutes. It has been proposed that the folding paths of large RNAs proceed through discrete intermediates but the nature of these intermediates is not known in most cases. Here, we describe our studies on the folding of the M1 RNA sub-unit of Escherichia coli RNase P. We performed kinetic footprinting studies of M1 RNA folding with the chemical footprinting reagent peroxynitrous acid to provide a detailed description of the folding pathway of RNase P RNA. Our results indicate that, in contrast to the Group I ribozyme, the M1 RNA folds into its catalytically active structure through the formation of two separately folded domains and that the folding of each proceeds through a discrete series of intermediates. Similar rates of folding were observed for regions believed to form the interface between the two domains. This observation is consistent with a kinetic trap which occurs by interaction of the domains during folding., (Copyright 2000 Academic Press.)

N-Hydroxypyridine-2(1H)-thione (N-HPT) has been studied as a photochemical source of hydroxyl radicals for use in photoinitiated nucleic acid footprinting experiments. Steady-state photolysis of dilute aqueous solutions of N-HPT at 350 nm in the presence of a 385 nucleotide (32)P-labeled RNA, the Tetrahymena L-21 ribozyme, resulted in cleavage of the RNA at nucleotide resolution. No cleavage of the RNA occurred in the absence of light or in the absence of N-HPT. Photolysis of the analogous pyridine lacking the N-hydroxyl group did not result in detectable amounts of RNA cleavage. The addition of RNA to preirradiated solutions of N-HPT gave no apparent RNA cleavage products, suggesting that the photoproducts of N-HPT do not result in RNA modification. Cleavage of RNA, upon photolysis in the presence of N-HPT, occurred in a sequence-independent fashion with double-stranded RNA being cleaved as efficiently as single-stranded RNA. Based on these observations, we conclude that photochemically generated diffusable hydroxyl radicals are responsible for the RNA cleavage. Experiments involving the photolysis of N-HPT in the presence of the Tetrahymena ribozyme and magnesium showed a magnesium-dependent protection from RNA cleavage due to formation of a folded RNA tertiary structure. The locations and amount of protection were identical to those observed in footprinting experiments performed with other hydroxyl radical sources. The presence of N-HPT had no effect on either the rate of folding or the catalytic activity of the folded RNA, indicating that this reagent does not disrupt RNA tertiary structure or otherwise affect activity. Thus, N-HPT is established as a new reagent for use in photoinitiated RNA footprinting experiments.

Following footprints to discover a path is easier with peroxynitrous acid. The folding of the Tetrahymena ribozyme was studied using this readily available reagent to generate hydroxyl radicals for kinetic footprinting studies. The different domains of the ribozyme appear to assemble at different rates following an ordered, hierarchical pathway (see scheme).

Large RNAs fold into complex structures which determine their biological activities. A full understanding of both RNA structure and dynamics will include the description of the pathways by which these structures are formed. Kinetic footprinting [Sclavi, B., et al. (1997) J. Mol. Biol. 266, 144-159] has been shown to be a powerful method for the study of dynamic processes involving RNA. Here we describe the use of a readily available reagent, peroxynitrous acid, as a kinetic footprinting tool for the study of RNA folding. Hydroxyl radicals generated from this reagent were used to footprint the Tetrahymena ribozyme during its magnesium-dependent folding-in agreement with synchroton X-ray footprinting [Sclavi, B., et al. (1998) Science 279, 1940-1943] and oligonucleotide/hybridization cleavage experiments [Zarrinkar, P. P., and Williamson, J. R. (1994) Science 265, 918-924], this work suggests an ordered, hierarchical folding pathway for the ribozyme. Several slow steps in the folding pathway were observed in the peroxynitrous acid footprinting, but none of these corresponded to the rate-determining step of folding. This suggests that the formation of the global, protected structure is followed by one or more slow local rearrangements to yield the final active structure. These studies illustrate the utility of peroxynitrous acid as a reagent for the elucidation of RNA folding pathways and the study of RNA dynamics.

We report here the first photo-chemical control of a ribozyme reaction by the site-specific modification of the 2'-hydroxyl nucleophile in the hammerhead system with a caging functionality. Rapid laser photolysis of the O-(2-nitrobenzyl) caging group initiates an efficient and accurate hammerhead-catalyzed cleavage of substrate RNA under native conditions. RNAs in which reactive functionalities or recognition elements are caged in this manner will be useful tools to probe RNA reactivity and dynamics.