Thomas Karl

Ribosomal proteins Rpl10 and Rps6 are potent regulators of yeast replicative life span.

The yeast ribosome is composed of two subunits, the large 60S subunit (LSU) and the small 40S subunit (SSU) and harbors 78 ribosomal proteins (RPs), 59 of which are encoded by duplicate genes. Recently, deletions of the LSU paralogs RPL31A and RPL6B were found to increase significantly yeast replicative life span (RLS). RPs Rpl10 and Rps6 are known translational regulators. Here, we report that heterozygosity for rpl10Delta but not for rpl25Delta, both LSU single copy RP genes, increased RLS by 24%. Deletion of the SSU RPS6B paralog, but not of the RPS6A paralog increased replicative life span robustly by 45%, while deletion of both the SSU RPS18A, and RPS18B paralogs increased RLS moderately, but significantly by 15%. Altering the gene dosage of RPL10 reduced the translating ribosome population, whereas deletion of the RPS6A, RPS6B, RPS18A, and RPS18B paralogs produced a large shift in free ribosomal subunit stoichiometry. We observed a reduction in growth rate in all deletion strains and reduced cell size in the SSU RPS6B, RPS6A, and RPS18B deletion strains. Thus, reduction of gene dosage of RP genes belonging to both the 60S and the 40S subunit affect lifespan, possibly altering the aging process by modulation of translation.

Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan

Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response.

Oxidative Stress in Fungi: Its Function in Signal Transduction, Interaction with Plant Hosts, and Lignocellulose Degradation

In this review article, we want to present an overview of oxidative stress in fungal cells in relation to signal transduction, interaction of fungi with plant hosts, and lignocellulose degradation. We will discuss external oxidative stress which may occur through the interaction with other microorganisms or plant hosts as well as internally generated oxidative stress, which can for instance originate from NADPH oxidases or “leaky” mitochondria and may be modulated by the peroxiredoxin system or by protein disulfide isomerases thus contributing to redox signaling. Analyzing redox signaling in fungi with the tools of molecular genetics is presently only in its beginning. However, it is already clear that redox signaling in fungal cells often is linked to cell differentiation (like the formation of perithecia), virulence (in plant pathogens), hyphal growth and the successful passage through the stationary phase.

Translational regulator RpL10p/Grc5p interacts physically and functionally with Sed1p, a dynamic component of the yeast cell surface

Biogenesis of an active ribosome complement and a dynamic cell surface complement are two major determinants of cellular growth. In yeast, the 60S ribosomal subunit protein RpL10p/Grc5p functions during successive stages in ribosome biogenesis, specifically rRNA processing, nucle(ol)ar preribosomal subunit assembly, nucleo‐cytoplasmic transport and cytoplasmic maturation of ribosomes. Here, we report that a two‐hybrid screen identified yeast genes SED1, ACS2 and PLB3 as encoding proteins physically interacting with both ribosomal RpL10p/Grc5p and its human homologue hRpL10p/QMp. SED1 encodes a differentially expressed cell wall protein which is proposed to be first transiently secreted to the plasma membrane as a GPI (glycosylated derivative of phosphoinositol)‐anchored form and to be then transferred to the glucan layer of the cell wall. Ectopic expression of SED1 rescues both the aberrant growth phenotype and the translation defect of grc5‐1ts temperature‐sensitive cells. Furthermore, we report that Sed1p associates with translating ribosomes suggesting a novel, cytoplasmic role for Sed1p. ACS2 encodes one of the two yeast acetyl‐CoA synthases and represents a key enzyme in one of several metabolic routes to produce acetyl‐CoA, which in turn is indispensable for lipid biosynthesis. PLB3 encodes a phospholipase, which is active in the breakdown of membrane lipids. Our results support the view that Grc5p/RpL10p links ribosome function to membrane turnover and cell surface biogenesis. Copyright

Protein signatures of oxidative stress response in a patient specific cell line model for autism

BackgroundKnown genetic variants can account for 10% to 20% of all cases with autism spectrum disorders (ASD). Overlapping cellular pathomechanisms common to neurons of the central nervous system (CNS) and in tissues of peripheral organs, such as immune dysregulation, oxidative stress and dysfunctions in mitochondrial and protein synthesis metabolism, were suggested to support the wide spectrum of ASD on unifying disease phenotype. Here, we studied in patient-derived lymphoblastoid cell lines (LCLs) how an ASD-specific mutation in ribosomal protein RPL10 (RPL10[H213Q]) generates a distinct protein signature. We compared the RPL10[H213Q] expression pattern to expression patterns derived from unrelated ASD patients without RPL10[H213Q] mutation. In addition, a yeast rpl10 deficiency model served in a proof-of-principle study to test for alterations in protein patterns in response to oxidative stress.MethodsProtein extracts of LCLs from patients, relatives and controls, as well as diploid yeast cells hemizygous for rpl10, were subjected to two-dimensional gel electrophoresis and differentially regulated spots were identified by mass spectrometry. Subsequently, Gene Ontology database (GO)-term enrichment and network analysis was performed to map the identified proteins into cellular pathways.ResultsThe protein signature generated by RPL10[H213Q] is a functionally related subset of the ASD-specific protein signature, sharing redox-sensitive elements in energy-, protein- and redox-metabolism. In yeast, rpl10 deficiency generates a specific protein signature, harboring components of pathways identified in both the RPL10[H213Q] subjects’ and the ASD patients’ set. Importantly, the rpl10 deficiency signature is a subset of the signature resulting from response of wild-type yeast to oxidative stress.ConclusionsRedox-sensitive protein signatures mapping into cellular pathways with pathophysiology in ASD have been identified in both LCLs carrying the ASD-specific mutation RPL10[H213Q] and LCLs from ASD patients without this mutation. At pathway levels, this redox-sensitive protein signature has also been identified in a yeast rpl10 deficiency and an oxidative stress model. These observations point to a common molecular pathomechanism in ASD, characterized in our study by dysregulation of redox balance. Importantly, this can be triggered by the known ASD-RPL10[H213Q] mutation or by yet unknown mutations of the ASD cohort that act upstream of RPL10 in differential expression of redox-sensitive proteins.

Specialized yeast ribosomes: a customized tool for selective mRNA translation.

Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered ‘specialized’ ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin β3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered.

A Novel Mutation in RPL10 (Ribosomal Protein L10) causes X‐linked Intellectual Disability, Cerebellar Hypoplasia and Spondylo‐Epiphyseal Dysplasia

RPL10 encodes ribosomal protein L10 (uL16), a highly conserved multifunctional component of the large ribosomal subunit, involved in ribosome biogenesis and function. Using X‐exome resequencing, we identified a novel missense mutation (c.191C>T; p.(A64V)) in the N‐terminal domain of the protein, in a family with two affected cousins presenting with X‐linked intellectual disability, cerebellar hypoplasia, and spondylo‐epiphyseal dysplasia (SED). We assessed the impact of the mutation on the translational capacity of the cell using yeast as model system. The mutation generates a functional ribosomal protein, able to complement the translational defects of a conditional lethal mutation of yeast rpl10. However, unlike previously reported mutations, this novel RPL10 missense mutation results in an increase in the actively translating ribosome population. Our results expand the mutational and clinical spectrum of RPL10 identifying a new genetic cause of SED and highlight the emerging role of ribosomal proteins in the pathogenesis of neurodevelopmental disorders.

Corrigendum: Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan

Corrigendum: Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan