Ema Kikovska

Eukaryotic RNase P RNA mediates cleavage in the absence of protein

The universally conserved ribonucleoprotein RNase P is involved in the processing of tRNA precursor transcripts. RNase P consists of one RNA and, depending on its origin, a variable number of protein subunits. Catalytic activity of the RNA moiety so far has been demonstrated only for bacterial and some archaeal RNase P RNAs but not for their eukaryotic counterparts. Here, we show that RNase P RNAs from humans and the lower eukaryote Giardia lamblia mediate cleavage of four tRNA precursors and a model RNA hairpin loop substrate in the absence of protein. Compared with bacterial RNase P RNA, the rate of cleavage (kobs) was five to six orders of magnitude lower, whereas the affinity for the substrate (appKd) was reduced ≈20- to 50-fold. We conclude that the RNA-based catalytic activity of RNase P has been preserved during evolution. This finding opens previously undescribed ways to study the role of the different proteins subunits of eukaryotic RNase P.

Protein Folding Activity of Ribosomal RNA Is a Selective Target of Two Unrelated Antiprion Drugs

Background 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases. Methodology/Principal Findings Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome. Conclusion/Significance 6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity.

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+1C+72 base pair at the end of the aminoacyl acceptor-stem whereas in tRNAGln G+1C+72 is replaced with U+1A+72. Here, we investigated RNase P RNA-mediated cleavage as a function of having G+1C+72 versus U+1A+72 in various substrate backgrounds, two full-size tRNA precursors (pre-tRNAGln and pre-tRNATyrSu3) and a model RNA hairpin substrate (pATSer). Our data showed that replacement of G+1C+72 with U+1A+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-tRNAGln and pre-tRNATyrSu3. 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 Mg2+ binding, most likely in its vicinity.

The naturally trans-acting ribozyme RNase P RNA has leadzyme properties.

Divalent metal ions promote hydrolysis of RNA backbones generating 5′OH and 2′;3′P as cleavage products. In these reactions, the neighboring 2′OH act as the nucleophile. RNA catalyzed reactions also require divalent metal ions and a number of different metal ions function in RNA mediated cleavage of RNA. In one case, the LZV leadzyme, it was shown that this catalytic RNA requires lead for catalysis. So far, none of the naturally isolated ribozymes have been demonstrated to use lead to activate the nucleophile. Here we provide evidence that RNase P RNA, a naturally trans-acting ribozyme, has leadzyme properties. But, in contrast to LZV RNA, RNase P RNA mediated cleavage promoted by Pb2+ results in 5′ phosphate and 3′OH as cleavage products. Based on our findings, we infer that Pb2+ activates H2O to act as the nucleophile and we identified residues both in the substrate and RNase P RNA that most likely influenced the positioning of Pb2+ at the cleavage site. Our data suggest that Pb2+ can promote cleavage of RNA by activating either an inner sphere H2O or a neighboring 2′OH to act as nucleophile.

Cleavage mediated by the P15 domain of bacterial RNase P RNA

Independently folded domains in RNAs frequently adopt identical tertiary structures regardless of whether they are in isolation or are part of larger RNA molecules. This is exemplified by the P15 domain in the RNA subunit (RPR) of the universally conserved endoribonuclease P, which is involved in the processing of tRNA precursors. One of its domains, encompassing the P15 loop, binds to the 3′-end of tRNA precursors resulting in the formation of the RCCA–RNase P RNA interaction (interacting residues underlined) in the bacterial RPR–substrate complex. The function of this interaction was hypothesized to anchor the substrate, expose the cleavage site and result in re-coordination of Mg2+ at the cleavage site. Here we show that small model-RNA molecules (~30 nt) carrying the P15-loop mediated cleavage at the canonical RNase P cleavage site with significantly reduced rates compared to cleavage with full-size RPR. These data provide further experimental evidence for our model that the P15 domain contributes to both substrate binding and catalysis. Our data raises intriguing evolutionary possibilities for ‘RNA-mediated’ cleavage of RNA.