Correlations of microRNA:microRNA expression patterns reveal insights into microRNA clusters and global microRNA expression patterns.

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.

Kinetic analysis of the M1 RNA folding pathway.

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.

Studies of RNA cleavage by photolysis of N-hydroxypyridine-2(1H)-thione. A new photochemical footprinting method.

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.

Characterization of the Tetrahymena ribozyme folding pathway using the kinetic footprinting reagent peroxynitrous acid.

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.

Caged RNA: photo-control of a ribozyme reaction.

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.