DNA and RNA contain, next to the four canonical nucleobases, a number of modified nucleosides that extend their chemical information content. RNA is particularly rich in modifications, which is obviously an adaptation to their highly complex and variable functions. In fact, the modified nucleosides and their chemical structures establish a second layer of information which is of central importance to the function of the RNA molecules. Also the chemical diversity of DNA is greater than originally thought. Next to the four canonical bases, the DNA of higher organisms contains a total of four epigenetic bases: m(5) dC, hm(5) dC, f(5) dC und ca(5) dC. While all cells of an organism contain the same genetic material, their vastly different function and properties inside complex higher organisms require the controlled silencing and activation of cell-type specific genes. The regulation of the underlying silencing and activation process requires an additional layer of epigenetic information, which is clearly linked to increased chemical diversity. This diversity is provided by the modified non-canonical nucleosides in both DNA and RNA.

The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer’s disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i 6 A) into 2-methylthio-N6-isopentenyladenosine (ms 2 i 6 A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.

Useful diversity: Quantification of modified tRNA nucleobases in different murine and porcine tissues reveals a tissue‐specific overall modification content. The modification content correlates with rates of protein synthesis in vitro, suggesting a direct link between tRNA modification levels and tissue‐specific translational efficiency.

The genetic system contains several levels of information. Firstly, the sequence of the canonical bases A, C, G, and T/U in DNA and RNA encodes amino acids through specific base triplets. Secondly, the methylation status of the cytosine base in DNA imprints epigenetic information into the genetic system, thereby contributing to the division of genes into active and inactive elements. Both information layers are chemically well investigated. Less is known about a putative third level of information associated with the chemical modification of RNA nucleobases. Although RNA, and in particular tRNA, is known to contain more than 100 different modified nucleosides, [1] the exact type of information added by base modification is largely unknown. A number of common modifications have been shown to improve the maintenance of the reading frame, [2] influence RNA stability, [3] and to be involved in proof-reading by tRNA synthetases. [4] Recently, it was discovered that the collective set of modified tRNA nucleosides is a regulated component of stress response and gives us a first hint that cells may actively adjust the modification pattern in response to external factors. [5] To learn more about the function of modified nucleobases we have investigated relationships between species by quantification of the modification content. By using a parallel systems-type approach we discovered that the collective set of modified bases is highly species-specific and linked to phylogeny. These data then enabled us to calculate a detailed phylogenetic tree that is consistent with those obtained from traditional data such as the homology of rRNA sequences, [6] conserved orthologous genes, [7] sequences of tRNA synthetases, [8] and tRNA-dependent amidotransferases.[9] The result shows that the set of base modifications is not universal, but rather a highly species-specific code under evolutionary selection to appropriately match base triplets with the corresponding cognate amino acids.For the study, we applied our recently developed LC-MSbased method for the quantification of modified nucleosides by using isotopically labeled standards.[10] For the parallel quantification we synthesized 18 tRNA modifications in both their natural and isotopically labeled forms (Scheme 1). A number of these nucleosides are present in the 3'-position to the anticodon in position 37, while others are distributed through the tRNA structure. The modifications studied are involved in a range of biological processes such as structural stabilization, codon binding, and translation initiation. [2a, 3, 10b, 11] With these tRNA nucleosides in hand, we analyzed the tRNA modification pattern of 16 species, so as to cover several branches of the phylogenetic tree. These species include five eukaryotes as well as five Gram-negative proteobacteria and five Gram-positive bacteria of the firmicutes. In addition we studied the bacterium Deinococcus radiodurans, which has a somewhat ambiguous classification. D. radiodurans is typically identified as Gram-positive, but ...