TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae.

Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously.

A post-labeling approach for the characterization and quantification of RNA modifications based on site-directed cleavage by DNAzymes.

Deoxyribozymes or DNAzymes are small DNA molecules with catalytic activity originating from in vitro selection experiments. Variants of the two most popular DNAzymes with RNase activity, the 10-23 DNAzyme and the 8-17 DNAzyme, promote efficient in vitro cleavage of the phosphodiester bond in at least 11 out of 16 possible dinucleotide permutations. Judicious choice of the sequences flanking the active core of the DNAzymes permits to direct cleavage activity with high sequence specificity. Here, the harnessing of these features for the analysis of RNA nucleotide modifications by a post-labeling approach is described in detail. DNAzymes are designed such that RNase cleavage is directed precisely to the 5' end of the nucleotide to be analyzed. Iterative complex formation of DNAzyme and RNA substrate and subsequent cleavage are performed by temperature cycling. The DNAzyme activity liberates the analyte nucleotide on the very 5'-end of an RNA fragment, whose hydroxyl group can be conveniently phosphorylated with (32)P. The labeled RNA is digested to mononucleotides, and analyzed by thin layer chromatography.

RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage.

Dnmt2 proteins are the most conserved members of the DNA methyltransferase enzyme family, but their substrate specificity and biological functions have been a subject of controversy. We show here that, in addition to tRNA(Asp-GTC), tRNA(Val-AAC) and tRNA(Gly-GCC) are also methylated by Dnmt2. Drosophila Dnmt2 mutants showed reduced viability under stress conditions, and Dnmt2 relocalized to stress granules following heat shock. Strikingly, stress-induced cleavage of tRNAs was Dnmt2-dependent, and Dnmt2-mediated methylation protected tRNAs against ribonuclease cleavage. These results uncover a novel biological function of Dnmt2-mediated tRNA methylation, and suggest a role for Dnmt2 enzymes during the biogenesis of tRNA-derived small RNAs.

Human DNMT2 methylates tRNA(Asp) molecules using a DNA methyltransferase-like catalytic mechanism.

Although their amino acid sequences and structure closely resemble DNA methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to function as RNA methyltransferases transferring a methyl group to the C5 position of C38 in tRNA(Asp). We observe that human DNMT2 methylates tRNA isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium discoideum. RNA extracted from wild type D. melanogaster was methylated to a lower degree, but in the case of Dictyostelium, there was no difference in the methylation of RNA isolated from wild-type and Dnmt2 knock-out strains. Methylation of in vitro transcribed tRNA(Asp) confirms it to be a target of DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA methyltransferase-like mechanism, because similar residues from motifs IV, VI, and VIII are involved in catalysis as identified in DNA methyltransferases. In addition, exchange of C292, which is located in a CFT motif conserved among Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2 represents the first example of an RNA methyltransferase using a DNA methyltransferase type of mechanism.

Use of DNAzymes for site-specific analysis of ribonucleotide modifications.

Post-transcriptional ribonucleotide modifications are widespread and abundant processes that have not been analyzed adequately due to the lack of appropriate detection methods. Here, two methods for the analysis of modified nucleotides in RNA are presented that are based on the quantitative and site-specific DNAzyme-mediated cleavage of the target RNA at or near the site of modification. Quantitative RNA cleavage is achieved by cycling the DNAzyme and its RNA substrate through repeated periods of heating and cooling. In a first approach, DNAzyme-directed cleavage directly 5' of the residue in question allows radioactive labeling of the newly freed 5'-OH. After complete enzymatic hydrolysis, the modification status can be assessed by two-dimensional thin layer chromatography. In a second approach, oligoribonucleotide fragments comprising the modification site are excised from the full-length RNA in an endonucleolytic fashion, using a tandem DNAzyme. The excised fragment is isolated by electrophoresis and submitted to further conventional analysis. These results establish DNAzymes as valuable tools for the site-specific and highly sensitive detection of ribonucleotide modifications.

Non-mammalian models for epigenetic analyses in cancer.

Many paradigms for our understanding of cancer etiology have been shaped in mammalian model systems. However, it has become evident that both genetic and epigenetic components actively influence the progression and severity of cancers. The complexity of epigenetic mechanisms in mammals has invigorated the use of non-mammalian model organisms in several research areas. Key contributions from this approach include (1) the in-depth characterization of epigenetic mechanisms and their interactions, resulting in an improved understanding of epigenetic pathways, (2) the establishment and refinement of techniques for genome-wide epigenetic profiling and (3) the discovery of novel epigenetic modifiers with potentially druggable enzymatic activities. Recent findings in all three areas will improve our understanding of epigenetic misregulation in cancer and facilitate the translation of basic research concepts into clinical applications.