Nsun5 methylates rRNA: 'specialized' ribosomes in stress resistance and aging



Over the past few years our research was focused on genomic and proteomic analyses of various primary human cells (Chang et al., 2005; Voglauer et al., 2005; Wieser et al. 2008) and CHO cell lines (Grillari et al., 2001). In order to functionally characterize some of the proteins involved in the regulation of cell proliferation, senescence and stress resistance, we started to characterize the novel protein SNEV as conserved splicing factor (Grillari et al., 2005) that extends cellular life span in dependence of ATM (Dellago et al., 2012), is involved in skin aging (Monteforte et al., 2016) and increases life span of D. melanogaster (Garschall et al., 2017).

Recently, we identified the protein Nsun5 as part of an evolutionarily conserved pathway regulating longevity (Schosserer et al., 2015). Thereby, Nsun5 methylates rRNA near the decoding center of the ribosomes and thus alters the recruitment of mRNAs during stress as observed using yeast as a genetic model system (Schosserer et al., 2015), supporting the hypothesis of “specialized ribosomes” (Xue and Barna, 2012).

Enzymatic characteristics of Nsun5 have not yet been established, but are necessary to fully understand its role in modulating the ribosome. A knock-out mouse model is already available in our lab. Since it confers increased stress resistance and alters ribosome function, this project will impact not only on research for healthy aging, but might also represent a novel target for engineering of cells from yeast to mammalian production systems in biotechnology, offering opportunities to engineer ribosome/mRNA pairs for high recombinant protein production.

Aims and methods.

We here propose to establish biochemical and genetic assays to characterize the enzymatic activity of human Nsun5 and its yeast homologue Rcm1 versus its substrate cytosin C3761 28S rRNA (human) and its ‘orthologous’ nucleotide C2278 of 25S rRNA (yeast). Therefore, we will produce recombinant human and yeast Nsun5 in yeast or in CHO cells in collaboration with BORTH and MATTANOVICH, purify it and perform in vitro methylation assays using radiolabelled SAM as methyl group donor and rRNA (precursor rRNA, full length and fragments) as substrates. In order to test for small molecules inhibiting Nsun5 activity, around 20 potential inhibitors have already been modelled in collaboration with OOSTENBRINK, and these will be tested in the biochemical assays for their potential to inhibit Nsun5 activity. As controls, other members of the Nsun RNA methylase family, especially Nsun2 will be used. Furthermore, the residues within the catalytic centre of Nsun5, especially the conserved cysteines, will be mutated site directedly in order to get first insights into the reaction mechanism.

In case sufficient Nsun5 protein is purified, we will also try to establish crystal structures in combination with its substrate in cooperation with OBINGER. In addition, (ii) we will establish combined bisulfite restriction analysis (COBRA) as a semiquantitative method to analyse the methylation degree of the C2278 site of 25S rRNA as well as human C3761 in 28S rRNA in analogy to COBRA used in testing for DNA methylation sites (Eads and Laird, 2002). This method can then be used to study the methylation degree of rRNA at specific sites in yeast and human cells under stress, aging, as well as in dependence of calorie restriction. Finally, we will also test the methylation degree of rRNA after exposure of cells to putative Nsun5 inhibitors. The compounds with the best Nsun5 inhibitory activity will then be tested for their effect on life span in C. elegans (WILSON).

Therefore, at the end of this project we will have characterized the enzymatic activity of Nsun5 in vitro and tested the inhibitory capacity of in silico modelled Nsun5 inhibitory compounds in the here developed medium throughput assay system. Finally, we will have confirmed that under various conditions cells respond by not-methylating the conserved cytosine in rRNA, which might be one mechanism to generate specialized ribosomes that selectively recruit specific mRNAs to be translated under changing environmental conditions.

Collaborations within this thesis will include OOSTENBRINK (modelling of Nsun5 inhibitory molecules), MATTANOVICH (recombinant expression of Nsun5 in yeast), BORTH (CHO cells) and WILSON (C. elegans).

Chang, M.W.F, Grillari, J., Mayrhofer, C., Fortschegger, K., Allmaier, G., Marzban, G., Katinger, H., Voglauer, R. (2005) Comparison of early passage, senescent and hTERT immortalized endothelial cells. Exp. Cell. Res. 309, 121-136. doi: 10.1016/ j.yexcr.2005.05.002
Dellago, H., Khan, A., Nussbacher, M., Gstraunthaler, A., Lämmermann, I., Schosserer, M., Mück, C., Anrather, D., Scheffold, A., Ammerer, G., Jansen-Dürr, P., Rudolph, K.L., Voglauer-Grillari, R., Grillari, J., Lammermann, I., Muck, C., Jansen-Durr, P. (2012) ATM-dependent phosphorylation of SNEVhPrp19/hPso4 is involved in extending cellular life span and suppression of apoptosis. Aging 4, 290-304. doi: 10.18632/aging.100452
Eads, C.A. and Laird, P.W. (2002) Combined bisulfite restriction analysis (COBRA). Methods Mol. Biol. 200, 71-85. doi: 10.1385/1-59259-182-5:071
Garschall, K., Dellago, H., Gáliková, M., Schosserer, M., Flatt, T., Grillari, J. (2017) Ubiquitous overexpression of the DNA repair factor dPrp19 reduces DNA damage and extends Drosophila life span. NPJ Aging Mech. Dis. 3, 5. doi: 10.1038/s41514-017-0005-z
Grillari, J., Fortschegger, K., Grabherr, R.M., Hohenwarter, O., Kunert, R., Katinger, H. (2001) Analysis of alterations in gene expression after amplification of recombinant genes in CHO cells. J. Biotechnol. 87, 59-65. doi: 10.16/S0168-1656(00)00431-4
Grillari, J., Ajuh, P., Stadler, G., Löscher, M., Voglauer, R., Ernst, W., Chusainow, J., Eisenhaber, F., Pokar, M., Fortschegger, K., Grey, M., Lamond, A.I., Katinger, H., Loscher, M. (2005) SNEV is an evolutionarily conserved splicing factor whose oligomerization is necessary for spliceosome assembly. Nucleic Acids Res. 33, 6868-6883. doi: 10.1093/nar/gki986
Hackl, M., Borth, N., Grillari, J. (2012) MiRNAs - pathway engineering of CHO cell factories that avoids translational burdening. Trends Biotechnol. 30, 405-406. doi: 10.1016/ j.tibtech.2012.05.002
Kondrashov, N., Pusic, A., Stumpf, C.R., Shimizu, K., Hsieh, A.C., Xue, S., Ishijima, J., Shiroishi, T., Barna, M. (2011) Ribosome-mediated specificityin Hox mRNA translation and vertebrate tissue patterning. Cell 145, 383-397. doi: 10.1016/j.cell.2011.03.028
Monteforte, R., Beilhack, G.F., Grausenburger, R., Mayerhofer, B., Bittner, R., Grillari-Voglauer, R., Sibilia, M., Dellago, H., Tschachler, E., Gruber, F., Grillari, J. (2016) SNEV P rp19/ PSO 4 deficiency increases PUVA-induced senescence in mouse skin. Exp. Dermatol. 25, 212-217. doi: 10.1111/exd.12910
Schosserer, M., Minois, N., Angerer, T.B., Amring, M., Dellago, H., Harreither, E., Calle-Perez, A., Pircher, A., Gerstl, M.P., Pfeifenberger, S., Brandl, C., Sonntagbauer, M., Kriegner, A., Linder, A., Weinhäusel, A., Mohr, T., Steiger, M., Mattanovich, D., Rinnerthaler, M., Karl, T., Sharma, S., Entian, K.-D., Kos, M., Breitenbach, M., Wilson, I.B.H., Polacek, N., Grillari-Voglauer, R., Breitenbach-Koller, L., Grillari, J. (2015) Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan. Nat. Commun. 6, 6158. doi: 10.1038/ncomms7158
Schuldt, A. (2011) Gene expression: personalized ribosomes. Nat. Rev. Mol. Cell Biol. 12, 344-345. doi: 10.1038/nrm3127
Voglauer, R., Grillari, J., Fortschegger, K., Wieser, M., Sterovsky, T., Gunsberg, P., Katinger, H., Pfragner, R. (2005) Establishment of human fibroma cell lines from a MEN1 patient by introduction of either hTERT or SV40 early region. Int. J. Oncol. 26, 961–970. doi: 10.3892/ijo.26.4.961
Weilner, S., Schraml, E., Redl, H., Grillari-Voglauer, R., Grillari, J. (2013) Secretion of microvesicular miRNAs in cellular and organismal aging. Exp. Gerontol. 48, 626-633. doi: 10.1016/j.exger.2012.11.017
Wieser, M., Stadler, G., Jennings, P., Streubel, B., Pfaller, W., Ambros, P., Riedl, C., Katinger, H., Grillari, J., Grillari-Voglauer, R. (2008) hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics. Am. J. Physiol. Ren. Physiol. 295, F1365-1375. doi: 10.1152/ajprenal.90405.2008
Xue, S. and Barna, M. (2012) Specialized ribosomes: a new frontier in gene regulation and organismal biology. Nat. Rev. Mol. Cell Biol. 13, 355-369. doi: 10.1038/nrm3359