2'-O-ribose methylation levels of ribosomal RNA distinguish different types of growth arrest in human dermal fibroblasts.

The 2'-O-methylation (2'-O-Me) of ribosomal RNA (rRNA) shows plasticity that is potentially associated with cell phenotypes. We used RiboMeth-seq profiling to reveal growth arrest-specific 2'-O-Me patterns in primary human dermal fibroblasts from three different donors. We exposed cells to hydrogen peroxide to induce cellular senescence and to high cell densities to promote quiescence by contact inhibition. We compared both modes of cell cycle arrest to proliferating cells and could indeed distinguish these conditions by their overall 2'-O-Me patterns. Methylation levels at a small fraction of sites showed plasticity and correlated with the expression of specific small nucleolar RNAs (snoRNAs) but not with expression of fibrillarin. Moreover, we observed subtle senescence-associated alterations in ribosome biogenesis. Knockdown of the snoRNA SNORD87, which acts as a guide for modification of a hypermethylated position in non-proliferating cells, was sufficient to boost cell proliferation. Conversely, depletion of SNORD88A, SNORD88B and SNORD88C, which act as guides for modification of a hypomethylated site, caused decreased proliferation without affecting global protein synthesis or apoptosis. Taken together, our findings provide evidence that rRNA modifications can be used to distinguish and potentially influence specific growth phenotypes of primary cells.

Loss of the ribosomal RNA methyltransferase NSUN5 impairs global protein synthesis and normal growth.

Modifications of ribosomal RNA expand the nucleotide repertoire and thereby contribute to ribosome heterogeneity and translational regulation of gene expression. One particular m5C modification of 25S ribosomal RNA, which is introduced by Rcm1p, was previously shown to modulate stress responses and lifespan in yeast and other small organisms. Here, we report that NSUN5 is the functional orthologue of Rcm1p, introducing m5C3782 into human and m5C3438 into mouse 28S ribosomal RNA. Haploinsufficiency of the NSUN5 gene in fibroblasts from William Beuren syndrome patients causes partial loss of this modification. The N-terminal domain of NSUN5 is required for targeting to nucleoli, while two evolutionary highly conserved cysteines mediate catalysis. Phenotypic consequences of NSUN5 deficiency in mammalian cells include decreased proliferation and size, which can be attributed to a reduction in total protein synthesis by altered ribosomes. Strikingly, Nsun5 knockout in mice causes decreased body weight and lean mass without alterations in food intake, as well as a trend towards reduced protein synthesis in several tissues. Together, our findings emphasize the importance of single RNA modifications for ribosome function and normal cellular and organismal physiology.

A Novel Caenorhabditis Elegans Proteinopathy Model Shows Changes in mRNA Translational Frameshifting During Aging.

BACKGROUND/AIMS: Regulation of mRNA translation is central to protein homeostasis and is optimized for speed and accuracy. Spontaneous recoding events occur virtually at any codon but at very low frequency and are commonly assumed to increase as the cell ages. METHODS: Here, we leveraged the polyglutamine(polyQ)-frameshifting model of huntingtin exon 1 with CAG repeat length in the pathological range (Htt51Q), which undergoes enhanced non-programmed translational -1 frameshifting. RESULTS: In body muscle cells of Caenorhabditis elegans, -1 frameshifting occured at the onset of expression of the zero-frame product, correlated with mRNA level of the non-frameshifted expression and formed aggregates correlated with reduced motility in C. elegans. Spontaneous frameshifting was modulated by IFG-1, the homologue of the nutrient-responsive eukaryotic initiation factor 4G (eIF4G), under normal growth conditions and NSUN-5, a conserved ribosomal RNA methyltransferase, under osmotic stress. CONCLUSION: Our results suggest that frameshifting and aggregation occur at even early stages of development and, because of their intrinsic stability, may persist and accelerate the onset of age-related proteinopathies.

The ribosomal RNA m5C methyltransferase NSUN-1 modulates healthspan and oogenesis in Caenorhabditis elegans.

Our knowledge about the repertoire of ribosomal RNA modifications and the enzymes responsible for installing them is constantly expanding. Previously, we reported that NSUN-5 is responsible for depositing m5C at position C2381 on the 26S rRNA in Caenorhabditis elegans. Here, we show that NSUN-1 is writing the second known 26S rRNA m5C at position C2982. Depletion of nsun-1 or nsun-5 improved thermotolerance and slightly increased locomotion at midlife, however, only soma-specific knockdown of nsun-1 extended lifespan. Moreover, soma-specific knockdown of nsun-1 reduced body size and impaired fecundity, suggesting non-cell-autonomous effects. While ribosome biogenesis and global protein synthesis were unaffected by nsun-1 depletion, translation of specific mRNAs was remodeled leading to reduced production of collagens, loss of structural integrity of the cuticle, and impaired barrier function. We conclude that loss of a single enzyme required for rRNA methylation has profound and highly specific effects on organismal development and physiology.