Ribo-Seq and RNA-Seq of TMA46 ( DFRP1) and GIR2 ( DFRP2) knockout yeast strains.

In eukaryotes, stalled and collided ribosomes are recognized by several conserved multicomponent systems, which either block protein synthesis in situ and resolve the collision locally, or trigger a general stress response. Yeast ribosome-binding GTPases RBG1 (DRG1 in mammals) and RBG2 (DRG2) form two distinct heterodimers with TMA46 (DFRP1) and GIR2 (DFRP2), respectively, both involved in mRNA translation. Accumulated evidence suggests that the dimers play partially redundant roles in elongation processivity and resolution of ribosome stalling and collision events, as well as in the regulation of GCN1-mediated signaling involved in ribosome-associated quality control (RQC). They also genetically interact with SLH1 (ASCC3) helicase, a key component of RQC trigger (RQT) complex disassembling collided ribosomes. Here, we present RNA-Seq and ribosome profiling (Ribo-Seq) data from S. cerevisiae strains with individual deletions of the TMA46 and GIR2 genes. Raw RNA-Seq and Ribo-Seq data as well as gene-level read counts are available in NCBI Gene Expression Omnibus (GEO) repository under GEO accession GSE185458 and GSE185286.

A Systematic Survey of Characteristic Features of Yeast Cell Death Triggered by External Factors.

Cell death in response to distinct stimuli can manifest different morphological traits. It also depends on various cell death signaling pathways, extensively characterized in higher eukaryotes but less so in microorganisms. The study of cell death in yeast, and specifically Saccharomyces cerevisiae, can potentially be productive for understanding cell death, since numerous killing stimuli have been characterized for this organism. Here, we systematized the literature on external treatments that kill yeast, and which contains at least minimal data on cell death mechanisms. Data from 707 papers from the 7000 obtained using keyword searches were used to create a reference table for filtering types of cell death according to commonly assayed parameters. This table provides a resource for orientation within the literature; however, it also highlights that the common view of similarity between non-necrotic death in yeast and apoptosis in mammals has not provided sufficient progress to create a clear classification of cell death types. Differences in experimental setups also prevent direct comparison between different stimuli. Thus, side-by-side comparisons of various cell death-inducing stimuli under comparable conditions using existing and novel markers that can differentiate between types of cell death seem like a promising direction for future studies.

The Pub1 and Upf1 Proteins Act in Concert to Protect Yeast from Toxicity of the [PSI⁺] Prion.

The [PSI⁺] nonsense-suppressor determinant of Saccharomyces cerevisiae is based on the formation of heritable amyloids of the Sup35 (eRF3) translation termination factor. [PSI⁺] amyloids have variants differing in amyloid structure and in the strength of the suppressor phenotype. The appearance of [PSI⁺], its propagation and manifestation depend primarily on chaperones. Besides chaperones, the Upf1/2/3, Siw14 and Arg82 proteins restrict [PSI⁺] formation, while Sla2 can prevent [PSI⁺] toxicity. Here, we identify two more non-chaperone proteins involved in [PSI⁺] detoxification. We show that simultaneous lack of the Pub1 and Upf1 proteins is lethal to cells harboring [PSI⁺] variants with a strong, but not with a weak, suppressor phenotype. This lethality is caused by excessive depletion of the Sup45 (eRF1) termination factor due to its sequestration into Sup35 polymers. We also show that Pub1 acts to restrict excessive Sup35 prion polymerization, while Upf1 interferes with Sup45 binding to Sup35 polymers. These data allow consideration of the Pub1 and Upf1 proteins as a novel [PSI⁺] detoxification system.

Ribosome-bound Pub1 modulates stop codon decoding during translation termination in yeast.

In eukaryotes, termination of translation is controlled by polypeptide chain release factors eRF1 and eRF3, of which the former recognizes nonsense codons, while the latter interacts with eRF1 and stimulates polypeptide release from the ribosome in a GTP- dependent manner, and ABCE1, which facilitates ribosome recycling. In this work, we demonstrate that Pub1, a yeast protein known to be involved in stress granule formation, regulation of gene expression, and organization of the tubulin cytoskeleton, also plays a role in translation termination. Pub1 was shown to bind to ribosomes independent of eRF1 and eRF3 and to interact with the N-terminal glutamine-/asparagine-rich prion domain of eRF3 via its short C-terminal glutamine-rich tract. High velocity sedimentation in sucrose gradient demonstrated that Pub1 was preferentially associated with heavy polysomes enriched with terminating ribosomes. Lack of Pub1 decreased efficiency of nonsense readthrough at a majority but not all tetranucleotide stop signals. This distinguishes Pub1 from most other known binding partners of the release factors which were shown to modulate readthrough of all nonsense codons uniformly. The obtained data show that Pub1 can act as an accessory translation factor involved in fine-tuning of translation termination.

Proteomic screening for amyloid proteins.

Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.

DNA aptamers detecting generic amyloid epitopes.

Amyloids are fibrillar protein aggregates resulting from non-covalent autocatalytic polymerization of various structurally and functionally unrelated proteins. Previously we have selected DNA aptamers, which bind specifically to the in vitro assembled amyloid fibrils of the yeast prionogenic protein Sup35. Here we show that such DNA aptamers can be used to detect SDS-insoluble amyloid aggregates of the Sup35 protein, and of some other amyloidogenic proteins, including mouse PrP, formed in yeast cells. The obtained data suggest that these aggregates and the Sup35 amyloid fibrils assembled in vitro possess common conformational epitopes recognizable by aptamers. The described DNA aptamers may be used for detection of various amyloid aggregates in yeast and, presumably, other organisms.

Purification and analysis of prion and amyloid aggregates.

Amyloids and prions represent aggregates of misfolded proteins, which consist of protein polymer fibrils with cross-beta sheet structure. Understanding of their occurrence and role is developing rapidly. Initially, they were found associated with mammalian diseases, mainly of neurodegenerative nature. Now they are known to relate to a range of non-disease phenomena in different species from mammals to lower eukaryotes. Uncovering new prion- and amyloid-related processes may be helped greatly by a procedure for purification of amyloid polymers. Studies of growth and propagation of these polymers require methods for determination of their size. Here, we describe such methods. They rely on the treatment with cold SDS or Sarcosyl detergents, which do not dissolve amyloids, but solubilize almost all non-amyloid complexes and associations between amyloid fibers. This allows purifying amyloids by centrifugation in the presence of these detergents. The size of amyloid polymers may be analyzed by electrophoresis in agarose gels containing SDS. Two procedures are described for determining the proportion between polymers and monomers of a particular protein using polyacrylamide gels.