Impaired tRNA Nuclear Export Links DNA Damage and Cell-Cycle Checkpoint

SummaryIn response to genotoxic stress, cells evoke a plethora of physiological responses collectively aimed at enhancing viability and maintaining the integrity of the genome. Here, we report that unspliced tRNA rapidly accumulates in the nuclei of yeast Saccharomyces cerevisiae after DNA damage. This response requires an intact MEC1- and RAD53-dependent signaling pathway that impedes the nuclear export of intron-containing tRNA via differential relocalization of the karyopherin Los1 to the cytoplasm. The accumulation of unspliced tRNA in the nucleus signals the activation of Gcn4 transcription factor, which, in turn, contributes to cell-cycle arrest in G1 in part by delaying accumulation of the cyclin Cln2. The regulated nucleocytoplasmic tRNA trafficking thus constitutes an integral physiological adaptation to DNA damage. These data further illustrate how signal-mediated crosstalk between distinct functional modules, namely, tRNA nucleocytoplasmic trafficking, protein synthesis, and checkpoint execution, allows for functional coupling of tRNA biogenesis and cell-cycle progression

SACCHARIS: an automated pipeline to streamline discovery of carbohydrate active enzyme activities within polyspecific families and de novo sequence datasets

Abstract Background Deposition of new genetic sequences in online databases is expanding at an unprecedented rate. As a result, sequence identification continues to outpace functional characterization of carbohydrate active enzymes (CAZymes). In this paradigm, the discovery of enzymes with novel functions is often hindered by high volumes of uncharacterized sequences particularly when the enzyme sequence belongs to a family that exhibits diverse functional specificities (i.e., polyspecificity). Therefore, to direct sequence-based discovery and characterization of new enzyme activities we have developed an automated in silico pipeline entitled: Sequence Analysis and Clustering of CarboHydrate Active enzymes for Rapid Informed prediction of Specificity (SACCHARIS). This pipeline streamlines the selection of uncharacterized sequences for discovery of new CAZyme or CBM specificity from families currently maintained on the CAZy website or within user-defined datasets. Results SACCHARIS was used to generate a phylogenetic tree of a GH43, a CAZyme family with defined subfamily designations. This analysis confirmed that large datasets can be organized into sequence clusters of manageable sizes that possess related functions. Seeding this tree with a GH43 sequence from Bacteroides dorei DSM 17855 (BdGH43b, revealed it partitioned as a single sequence within the tree. This pattern was consistent with it possessing a unique enzyme activity for GH43 as BdGH43b is the first described α-glucanase described for this family. The capacity of SACCHARIS to extract and cluster characterized carbohydrate binding module sequences was demonstrated using family 6 CBMs (i.e., CBM6s). This CBM family displays a polyspecific ligand binding profile and contains many structurally determined members. Using SACCHARIS to identify a cluster of divergent sequences, a CBM6 sequence from a unique clade was demonstrated to bind yeast mannan, which represents the first description of an α-mannan binding CBM. Additionally, we have performed a CAZome analysis of an in-house sequenced bacterial genome and a comparative analysis of B. thetaiotaomicron VPI-5482 and B. thetaiotaomicron 7330, to demonstrate that SACCHARIS can generate “CAZome fingerprints”, which differentiate between the saccharolytic potential of two related strains in silico. Conclusions Establishing sequence-function and sequence-structure relationships in polyspecific CAZyme families are promising approaches for streamlining enzyme discovery. SACCHARIS facilitates this process by embedding CAZyme and CBM family trees generated from biochemically to structurally characterized sequences, with protein sequences that have unknown functions. In addition, these trees can be integrated with user-defined datasets (e.g., genomics, metagenomics, and transcriptomics) to inform experimental characterization of new CAZymes or CBMs not currently curated, and for researchers to compare differential sequence patterns between entire CAZomes. In this light, SACCHARIS provides an in silico tool that can be tailored for enzyme bioprospecting in datasets of increasing complexity and for diverse applications in glycobiotechnology

A Genome Scale Screen for Mutants with Delayed Exit from Mitosis: Ire1-Independent Induction of Autophagy Integrates ER Homeostasis into Mitotic Lifespan.

Proliferating eukaryotic cells undergo a finite number of cell divisions before irreversibly exiting mitosis. Yet pathways that normally limit the number of cell divisions remain poorly characterized. Here we describe a screen of a collection of 3762 single gene mutants in the yeast Saccharomyces cerevisiae, accounting for 2/3 of annotated yeast ORFs, to search for mutants that undergo an atypically high number of cell divisions. Many of the potential longevity genes map to cellular processes not previously implicated in mitotic senescence, suggesting that regulatory mechanisms governing mitotic exit may be broader than currently anticipated. We focused on an ER-Golgi gene cluster isolated in this screen to determine how these ubiquitous organelles integrate into mitotic longevity. We report that a chronic increase in ER protein load signals an expansion in the assembly of autophagosomes in an Ire1-independent manner, accelerates trafficking of high molecular weight protein aggregates from the cytoplasm to the vacuoles, and leads to a profound enhancement of daughter cell production. We demonstrate that this catabolic network is evolutionarily conserved, as it also extends reproductive lifespan in the nematode Caenorhabditis elegans. Our data provide evidence that catabolism of protein aggregates, a natural byproduct of high protein synthesis and turn over in dividing cells, is among the drivers of mitotic longevity in eukaryotes

Rapid Nuclear Exclusion of Hcm1 in Aging Saccharomyces cerevisiae Leads to Vacuolar Alkalization and Replicative Senescence

The yeast, Saccharomyces cerevisiae, like other higher eukaryotes, undergo a finite number of cell divisions before exiting the cell cycle due to the effects of aging. Here, we show that yeast aging begins with the nuclear exclusion of Hcm1 in young cells, resulting in loss of acidic vacuoles. Autophagy is required for healthy aging in yeast, with proteins targeted for turnover by autophagy directed to the vacuole. Consistent with this, vacuolar acidity is necessary for vacuolar function and yeast longevity. Using yeast genetics and immunofluorescence microscopy, we confirm that vacuolar acidity plays a critical role in cell health and lifespan, and is potentially maintained by a series of Forkhead Box (Fox) transcription factors. An interconnected transcriptional network involving the Fox proteins (Fkh1, Fkh2 and Hcm1) are required for transcription of v-ATPase subunits and vacuolar acidity. As cells age, Hcm1 is rapidly excluded from the nucleus in young cells, blocking the expression of Hcm1 targets (Fkh1 and Fkh2), leading to loss of v-ATPase gene expression, reduced vacuolar acidification, increased α-syn-GFP vacuolar accumulation, and finally, diminished replicative lifespan (RLS). Loss of vacuolar acidity occurs about the same time as Hcm1 nuclear exclusion and is conserved; we have recently demonstrated that lysosomal alkalization similarly contributes to aging in C. elegans following a transition from progeny producing to post-reproductive life. Our data points to a molecular mechanism regulating vacuolar acidity that signals the end of RLS when acidification is lost

Enhanced clearance of protein aggregates in yeast and worm <i>rer1</i> mutants.

<p><b>A.</b> Live cell imaging of α-syn-GFP after a 4 hr induction in 0.2% galactose. TM (0.02 μg/ml) was added to the cultures after an initial induction in galactose and cells were imaged 2 hours later. In a marked contrast to untreated cells, the GFP foci were largely vacuolar in <i>rer1Δ</i> or wild type cells treated with TM (arrows). <b>B.</b> GFP immunoblot in whole cell lysates prepared from cells in (<b>A)</b>. The intravacuolar proteolysis of α-syn-GFP generates the transiently stable GFP moiety detected as a discrete fragment in SDS-PAGE. <b>C.</b> Fluorescent images of transgenic hermaphrodites stably expressing α-syn-GFP from a body wall muscle promoter (<i>Punc-</i>_<i>54</i>::<i>α-syn</i>::<i>GFP</i>). Arrows denote a subset of the α-syn-GFP foci. Worms co-expressing heat shock protein torsinA (<i>tor-2</i>) in the same cells (<i>P</i>_{<i>unc-54</i>}::<i>tor-2</i>) served as a positive experimental control. The average number of α-syn-GFP foci per worm scored from digitized Z-stacked images ± s.e.m are plotted in <b>D</b> (n >20).</p

ER stress extends reproductive lifespan in yeast and worms.

<p><b>A.</b> Mitotic lifespan of wild type yeast grown on YPD supplemented with the indicated concentrations of TM. Mean mitotic lifespans for WT (18.5) and cells treated with TM at 0.005 (24.5) and 0.02 (27.5) μg/ml were statistically significant (p-values<0.01). <b>B.</b> Reproductive lifespan in worms with or without dietary supplementation with TM. <b>C.</b> Live cell imaging of GFP-Rer1 in wild type yeast and <i>ire1Δ</i> mutants treated with DMSO or after a 2 hr exposure to 0.02 μg/ml TM. (Box) In tandem with inducing canonical UPR, ER stress increases trafficking from ER to Golgi, reflected in the Golgi redistribution of GFP-Rer1 in cells treated with TM. Conversely, deleting <i>RER1</i> increases ER load denoted by the high basal UPR in <i>rer1Δ</i> mutants. <b>D</b>. Mitotic lifespan of WT (16.5 days) and isogenic <i>rer1∆</i> (26.5), <i>ire1∆</i> (16.5) and <i>rer1∆ ire1∆</i> (24.5) mutants at 30°C. Differences between WT mean mitotic lifespan were significant for <i>rer1∆</i> and <i>rer1∆ ire1∆</i> mutants (p-values<0.001).</p

A genome scale screen for isolating mutants with extended mitotic lifespan in the yeast <i>S</i>. <i>cerevisiae</i>.

<p><b>A.</b> The screen work flow. The design rationale is detailed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005429#pgen.1005429.s001" target="_blank">S1 Fig</a>. <b>B.</b> Cy5:Cy3 signal ratios of mutants maintained in a dividing state for 16 days. Ranked values were log₂ normalized and projected. Of the starting collection of 3762, 52 mutants that maintained negative log₂ Cy5:Cy3 signal ratios at both day 6 and day 16 and displayed signal ratios <-2.3 at day 16 were classified as potentially long-lived (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005429#pgen.1005429.s003" target="_blank">S3 Fig</a>). <b>C.</b> Broad functional clustering of the putative longevity genes isolated in this screen using GO Ontology. Genes that function in protein modification and trafficking across the ER-Golgi network are outlined.</p