MMI1 (YKL056c, TMA19), the yeast orthologue of the translationally controlled tumor protein (TCTP) has apoptotic functions and interacts with both microtubules and mitochondria.

The yeast orthologue of mammalian TCTP is here proposed to be named Mmi1p (microtubule and mitochondria interacting protein). This protein displays about 50% amino acid sequence identity with its most distantly related orthologs in higher organisms and therefore probably belongs to a small class of yeast proteins which have housekeeping but so far incompletely known functions needed for every eukaryotic cell. Previous investigations of the protein in both higher cells and yeast revealed that it is highly expressed during active growth, but transcriptionally down-regulated in several kinds of stress situations including starvation stress. In human cells, TCTP presumably has anti-apoptotic functions as it binds to Bcl-XL in vivo. TCTP of higher cells was also shown to interact with the translational machinery. It has acquired an additional function in the mammalian immune system, as it is identical with the histamine releasing factor. Here, we show that in S. cerevisiae induction of apoptosis by mild oxidative stress, replicative ageing or mutation of cdc48 leads to translocation of Mmi1p from the cytoplasm to the mitochondria. Mmi1p is stably but reversibly attached to the outer surface of the mitochondria and can be removed by digestion with proteinase K. Glutathionylation of Mmi1p, which is also induced by oxidants, is not a prerequisite or signal for translocation as shown by replacing the only cysteine of Mmi1p by serine. Mmi1p probably interacts with yeast microtubules as deletion of the gene confers sensitivity to benomyl. Conversely, the deletion mutant displays resistance to hydrogen peroxide stress and shows a small but significant elongation of the mother cell-specific lifespan. Our results so far indicate that Mmi1p is one of the few proteins establishing a functional link between microtubules and mitochondria which may be needed for correct localization of mitochondria during cell division

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.Publisher PDFPeer reviewe

Drug Development for Target Ribosomal Protein rpL35/uL29 for Repair of LAMB3R635X in Rare Skin Disease Epidermolysis Bullosa

Introduction: Epidermolysis bullosa (EB) describes a family of rare genetic blistering skin disorders. Various subtypes are clinically and genetically heterogeneous, and a lethal postpartum form of EB is the generalized severe junctional EB (gs-JEB). gs-JEB is mainly caused by premature termination codon (PTC) mutations in the skin anchor protein LAMB3 (laminin subunit beta-3) gene. The ribosome in majority of translational reads of LAMB3PTC mRNA aborts protein synthesis at the PTC signal, with production of a truncated, nonfunctional protein. This leaves an endogenous readthrough mechanism needed for production of functional full-length Lamb3 protein albeit at insufficient levels. Here, we report on the development of drugs targeting ribosomal protein L35 (rpL35), a ribosomal modifier for customized increase in production of full-length Lamb3 protein from a LAMB3PTC mRNA. Methods: Molecular docking studies were employed to identify small molecules binding to human rpL35. Molecular determinants of small molecule binding to rpL35 were further characterized by titration of the protein with these ligands as monitored by nuclear magnetic resonance (NMR) spectroscopy in solution. Changes in NMR chemical shifts were used to map the docking sites for small molecules onto the 3D structure of the rpL35. Results: Molecular docking studies identified 2 FDA-approved drugs, atazanavir and artesunate, as candidate small-molecule binders of rpL35. Molecular interaction studies predicted several binding clusters for both compounds scattered throughout the rpL35 structure. NMR titration studies identified the amino acids participating in the ligand interaction. Combining docking predictions for atazanavir and artesunate with rpL35 and NMR analysis of rpL35 ligand interaction, one binding cluster located near the N-terminus of rpL35 was identified. In this region, the nonidentical binding sites for atazanavir and artesunate overlap and are accessible when rpL35 is integrated in its natural ribosomal environment. Conclusion: Atazanavir and artesunate were identified as candidate compounds binding to ribosomal protein rpL35 and may now be tested for their potential to trigger a rpL35 ribosomal switch to increase production of full-length Lamb3 protein from a LAMB3PTC mRNA for targeted systemic therapy in treating gs-JEB

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 ?3 (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

The mitochondrial ribosomal of the large subunit, afo1p, determines cellular longevity through mitochondrial back-signaling via TOR1

Abstract: Yeast mother cell‐specific aging constitutes a model of replicative aging as it occurs in stem cell populations ofhigher eukaryotes. Here, we present a new long‐lived yeast deletion mutation, afo1 (for aging factor one), that confers a60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of themitochondrial ribosome. Double mutant experiments indicate that the longevity‐increasing action of the afo1 mutation isindependent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth‐controllingtranscription factor Sfp1p. In their final cell cycle, the long‐lived mutant cells do show the phenotypes of yeast apoptosisindicating that the longevity of the mutant is not caused by an inaility to undergo programmed cell death. Furthermore,the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxicalincrease in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1and fob1 shows that the longevity phenotype of afo1 is independen of the formation of ERCs (ribosomal DNA minicircles).AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging

The mitochondrial ribosomal of the large subunit, afo1p, determines cellular longevity through mitochondrial back-signaling via TOR1

Abstract: Yeast mother cell‐specific aging constitutes a model of replicative aging as it occurs in stem cell populations ofhigher eukaryotes. Here, we present a new long‐lived yeast deletion mutation, afo1 (for aging factor one), that confers a60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of themitochondrial ribosome. Double mutant experiments indicate that the longevity‐increasing action of the afo1 mutation isindependent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth‐controllingtranscription factor Sfp1p. In their final cell cycle, the long‐lived mutant cells do show the phenotypes of yeast apoptosisindicating that the longevity of the mutant is not caused by an inaility to undergo programmed cell death. Furthermore,the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxicalincrease in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1and fob1 shows that the longevity phenotype of afo1 is independen of the formation of ERCs (ribosomal DNA minicircles).AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging