Substrate discrimination in RNase P RNA-mediated cleavage: importance of the structural environment of the RNase P cleavage site

Like the translational elongation factor EF-Tu, RNase P interacts with a large number of substrates where RNase P with its RNA subunit generates tRNAs with matured 5′ termini by cleaving tRNA precursors immediately 5′ of the residue at +1, i.e. at the position that corresponds to the first residue in tRNA. Most tRNAs carry a G(+1)C(+72) base pair at the end of the aminoacyl acceptor-stem whereas in tRNA(Gln) G(+1)C(+72) is replaced with U(+1)A(+72). Here, we investigated RNase P RNA-mediated cleavage as a function of having G(+1)C(+72) versus U(+1)A(+72) in various substrate backgrounds, two full-size tRNA precursors (pre-tRNA(Gln) and pre-tRNA(Tyr)Su3) and a model RNA hairpin substrate (pATSer). Our data showed that replacement of G(+1)C(+72) with U(+1)A(+72) influenced ground state binding, cleavage efficiency under multiple and single turnover conditions in a substrate-dependent manner. Interestingly, we observed differences both in ground state binding and rate of cleavage comparing two full-size tRNA precursors, pre-tRNA(Gln) and pre-tRNA(Tyr)Su3. These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step. In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA. Finally, we provide evidence that the structural architecture influences Mg(2+) binding, most likely in its vicinity

Metal ion cooperativity in Escherichia coli RNase P RNA

RNase P is an essential ribonuclease responsible for removal of the 5’ leader of tRNA precursors. Bacterial RNase P consists of an RNA subunit and a small basic protein. The catalytic activity is associated with the RNA subunit, i.e. bacterial RNase P RNA is a ribozyme. The protein subunit is, however, essential for activity in vivo. RNase P RNA, as well as the holoenzyme, requires the presence of divalent metal ions for activity. The aim of this thesis was to increase our understanding of the catalytic mechanism of RNase P RNA mediated cleavage. The importance of the nucleotides close to the cleavage site and the roles of divalent metal ions in RNase P RNA-catalyzed reaction were investigated. Escherichia coli RNase P RNA (M1 RNA) was used as a model system. It was shown that different metal ions have differential effects on cleavage site recognition. Cleavage activity was rescued by mixing metal ions that do not promote cleavage activity by themselves. This suggests that efficient and correct cleavage is the result of metal ion cooperativity in the RNase P RNA-mediated cleavage reaction. The results suggested that one of the metal ions involved in this cooperativity is positioned in the vicinity of a well-known interaction between RNase P RNA and its substrate. Based on my studies on how different metal ions bind to RNA and influence its activity we raise the interesting possibility that the activity of biocatalysts that depend on RNA for activity are up- or downregulated depending on the intracellular concentrations of the bulk biological metal ions Mg2+ and Ca2+. The nucleotides upstream of the cleavage site in the substrate were found to influence the cleavage efficiency. This was not exclusively due to intermolecular base pairing within the substrate but also dependent on the identities of the nucleotides at position –2 and –1. The strength of the base pair at position –1/+73 was demonstrated to affect cleavage efficiency. These observations are in keeping with previous suggestion that the nucleotides close to the cleavage site are important for RNase P cleavage. We conclude that the residue at -1 is a positive determinant for cleavage by RNase P. Hence, my studies extend our understanding of the RNase P cleavage site recognition process

Cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage

To monitor functionally important metal ions and possible cross talk in RNase P RNA mediated cleavage we studied cleavage of substrates, where the 2′OH at the RNase P cleavage site (at −1) and/or at position +73 had been replaced with a 2′ amino group (or 2′H). Our data showed that the presence of 2′ modifications at these positions affected cleavage site recognition, ground state binding of substrate and/or rate of cleavage. Cleavage of 2′ amino substituted substrates at different pH showed that substitution of Mg(2+) by Mn(2+) (or Ca(2+)), identity of residues at and near the cleavage site, and addition of C5 protein influenced the frequency of miscleavage at −1 (cleavage at the correct site is referred to as +1). From this we infer that these findings point at effects mediated by protonation/deprotonation of the 2′ amino group, i.e. an altered charge distribution, at the site of cleavage. Moreover, our data suggested that the structural architecture of the interaction between the 3′ end of the substrate and RNase P RNA influence the charge distribution at the cleavage site as well as the rate of cleavage under conditions where the chemistry is suggested to be rate limiting. Thus, these data provide evidence for cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage. We discuss the role metal ions might play in this cross talk and the likelihood that at least one functionally important metal ion is positioned in the vicinity of, and use the 2′OH at the cleavage site as an inner or outer sphere ligand

Illustration of the ‘A–RNase P RNA interaction’ (interacting residues underlined) and the interaction between A248 and residue −1 in the substrate, the ‘/N interaction’ for details see the text and (,), and references therein

<p><b>Copyright information:</b></p><p>Taken from "Substrate discrimination in RNase P RNA-mediated cleavage: importance of the structural environment of the RNase P cleavage site"</p><p>Nucleic Acids Research 2005;33(6):2012-2021.</p><p>Published online 7 Apr 2005</p><p>PMCID:PMC1074746.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The residues indicated in red correspond to those that were replaced in this report. The residues in red circles show the ‘+73/294 interaction’ while the arrow mark the canonical RNase P cleavage site at +1. In the case of the C variants, C is inferred to base pair with G in the absence of RNase P RNA. A and B (in gray circles) represent Mg ions that have been suggested to be positioned at and in the vicinity of the cleavage site [() and references therein] while C is an additional ion positioned in the P15-loop. The 2′-OH at the −1 position has been suggested coordinating Mg at the cleavage site, and/or interacting with RNase P RNA (,,,,,,)

Reproducibility of high-throughput mRNA and small RNA sequencing across laboratories

RNA sequencing is an increasingly popular technology for genome-wide analysis of transcript sequence and abundance. However, understanding of the sources of technical and interlaboratory variation is still limited. To address this, the GEUVADIS consortium sequenced mRNAs and small RNAs of lymphoblastoid cell lines of 465 individuals in seven sequencing centers, with a large number of replicates. The variation between laboratories appeared to be considerably smaller than the already limited biological variation. Laboratory effects were mainly seen in differences in insert size and GC content and could be adequately corrected for. In small-RNA sequencing, the microRNA (miRNA) content differed widely between samples owing to competitive sequencing of rRNA fragments. This did not affect relative quantification of miRNAs. We conclude that distributing RNA sequencing among different laboratories is feasible, given proper standardization and randomization procedures. We provide a set of quality measures and guidelines for assessing technical biases in RNA-seq data