Combined tRNA modification defects impair protein homeostasis and synthesis of the yeast prion protein Rnq1

Modified nucleosides in tRNA anticodon loops such as 5-methoxy-carbonyl-methyl-2-thiouridine (mcm⁵s²U) and pseuduridine (Ψ) are thought to be required for an efficient decoding process. In Saccharomyces cerevisiae, the simultaneous presence of mcm⁵s²U and Ψ38 in tRNA^Gln_UUG was shown to mediate efficient synthesis of the Q/N rich [PIN⁺] prion forming protein Rnq1.¹ Klassen R, Ciftci A, Johanna Funk J, Bruch A, Butter F, Schaffrath R. tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast. Nucleic Acids Res 2016; 44(22):10946-959. pii: gkw705; PMID:27496282; http://dx.doi.org/10.1093/nar/gkw705[Crossref], [PubMed], [Web of Science ®] [Google Scholar] In the absence of these two tRNA modifications, higher than normal levels of hypomodified tRNA^Gln_UUG, but not its isoacceptor tRNA^Gln_CUG can restore Rnq1 synthesis. Moroever, tRNA overexpression rescues pleiotropic phenotypes that associate with loss of mcm⁵s²U and Ψ38 formation. Notably, combined absence of different tRNA modifications are shown to induce the formation of protein aggregates which likely mediate severe cytological abnormalities, including cytokinesis and nuclear segregation defects. In support of this, overexpression of the aggregating polyQ protein Htt103Q, but not its non-aggregating variant Htt25Q phenocopies these cytological abnormalities, most pronouncedly in deg1 single mutants lacking Ψ38 alone. It is concluded that slow decoding of particular codons induces defects in protein homeostasis that interfere with key steps in cytokinesis and nuclear segregation.     

Solution structure of the yeast ubiquitin-like modifier protein Hub1

abbreviationsUBL, ubiquitin-like modifier; Saccharomyces cerevisiae, S. cerevisiae; Eschericia coli, E. coli; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy; TOCSY, total correlated spectroscopy.     

Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1

The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1-151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1-85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.     

Amyloid properties of the yeast cell wall protein Toh1 and its interaction with prion proteins Rnq1 and Sup35

Amyloids are non-branching fibrils that are composed of stacked monomers stabilized by intermolecular β-sheets. Some amyloids are associated with incurable diseases, whereas others, functional amyloids, regulate different vital processes. The prevalence and significance of functional amyloids in wildlife are still poorly understood. In recent years, by applying new approach of large-scale proteome screening, a number of novel candidate amyloids were identified in the yeast Saccharomyces cerevisiae, many of which are localized in the yeast cell wall. In this work, we showed that one of these proteins, Toh1, possess amyloid properties. The Toh1-YFP hybrid protein forms detergent-resistant aggregates in the yeast cells while being expressed under its own P_TOH1 or inducible P_CUP1 promoter. Using bacterial system for generation of extracellular amyloid aggregates C-DAG, we demonstrated that the N-terminal Toh1 fragment, containing amyloidogenic regions predicted in silico, binds Congo Red dye, manifests ‘apple-green’ birefringence when examined between crossed polarizers, and forms amyloid-like fibrillar aggregates visualized by TEM. We have established that the Toh1(20–365)-YFP hybrid protein fluorescent aggregates are co-localized with a high frequency with Rnq1C-CFP and Sup35NM-CFP aggregates in the yeast cells containing [PIN⁺] and [PSI⁺] prions, and physical interaction of these aggregated proteins was confirmed by FRET. This is one of a few known cases of physical interaction of non-Q/N-rich amyloid-like protein and Q/N-rich amyloids, suggesting that interaction of different amyloid proteins may be determined not only by similarity of their primary structures but also by similarity of their secondary structures and of conformational folds.     

SUMO: a ubiquitin-like protein modifier

SUMO (Small Ubiquitin-related Modifier) is a small protein that covalently attaches to a lysine residue of another protein in a reversible fashion. SUMO attachment to its substrate proteins causes changes in the localization, activity, or binding partners of the substrate. SUMO has been shown to play a role in a multitude of processes; these include chromosome segregation, cell cycle progression, and DNA damage recovery. Defects in the SUMO pathway have been demonstrated to affect tumorigenesis and the inflammatory response as well as other human diseases.     

Genomic patterns of DNA methylation: targets and function of an epigenetic mark

Methylation of cytosines can mediate epigenetic gene silencing and is the only known DNA modification in eukaryotes. Recent efforts to map DNA methylation across mammalian genomes revealed limited DNA methylation at regulatory regions but widespread methylation in intergenic regions and repeats. This is consistent with the idea that hypermethylation is the default epigenetic state and serves in maintaining genome integrity. DNA methylation patterns at regulatory regions are generally stable, but a minor subset of regulatory regions show variable DNA methylation between cell types, suggesting an additional dynamic component. Such promoter de novo methylation might be involved in the maintenance rather than the initiation of silencing of defined genes during development. How frequently such dynamic methylation occurs, its biological relevance and the pathways involved deserve investigation.     

Fission yeast pkl1 is a kinesin-related protein involved in mitotic spindle function.

We have used anti-peptide antibodies raised against highly conserved regions of the kinesin motor domain to identify kinesin-related proteins in the fission yeast Schizosaccharomyces pombe. Here we report the identification of a new kinesin-related protein, which we have named pkl1. Sequence homology and domain organization place pkl1 in the Kar3/ncd subfamily of kinesin-related proteins. Bacterially expressed pkl1 fusion proteins display microtubule-stimulated ATPase activity, nucleotide-sensitive binding, and bundling of microtubules. Immunofluorescence studies with affinity-purified antibodies indicate that the pkl1 protein localizes to the nucleus and the mitotic spindle. Pkl1 null mutants are viable but have increased sensitivity to microtubule-disrupting drugs. Disruption of pkl1+ suppresses mutations in another kinesin-related protein, cut7, which is known to act in the spindle. Overexpression of pkl1 to very high levels causes a similar phenotype to that seen in cut7 mutants: V-shaped and star-shaped microtubule structures are observed, which we interpret to be spindles with unseparated spindle poles. These observations suggest that pkl1 and cut7 provide opposing forces in the spindle. We propose that pkl1 functions as a microtubule-dependent motor that is involved in microtubule organization in the mitotic spindle.     

A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting

Members of the Mx protein family promote interferon-inducible resistance to viral infection in mammals and act by unknown mechanisms. We identified an Mx-like protein in yeast and present genetic evidence for its cellular function. This protein, the VPS1 product, is essential for vacuolar protein sorting, normal organization of intracellular membranes, and growth at high temperature, implying that Mx-like proteins are engaged in fundamental cellular processes in eukaryotes. Vps1p contains a tripartite GTP binding motif, which suggests that binding to GTP is essential to its role in protein sorting. Vps1p-specific antibody labels punctate cytoplasmic structures that condense to larger structures in a Golgi-accumulating sec7 mutant; thus, Vps1p may associate with an intermediate organelle of the secretory pathway.     

Chronic fluid flow is an environmental modifier of renal epithelial function

Although solitary or sensory cilia are present in most cells of the body and their existence has been known since the sixties, very little is been known about their functions. One suspected function is fluid flow sensing- physical bending of cilia produces an influx of Ca(++), which can then result in a variety of activated signaling pathways. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a progressive disease, typically appearing in the 5(th) decade of life and is one of the most common monogenetic inherited human diseases, affecting approximately 600,000 people in the United States. Because ADPKD is a slowly progressing disease, I asked how fluid flow may act, via the primary cilium, to alter epithelial physiology during the course of cell turnover. I performed an experiment to determine under what conditions fluid flow can result in a change of function of renal epithelial tissue. A wildtype epithelial cell line derived the cortical collecting duct of a heterozygous offspring of the Immortomouse (Charles River Laboratory) was selected as our model system. Gentle orbital shaking was used to induce physiologically relevant fluid flow, and periodic measurements of the transepithelial Sodium current were performed. At the conclusion of the experiment, mechanosensitive proteins of interest were visualized by immunostaining. I found that fluid flow, in itself, modifies the transepithelial sodium current, cell proliferation, and the actin cytoskeleton. These results significantly impact the understanding of both the mechanosensation function of primary cilia as well as the understanding of ADPKD disease progression.     

EWSR1, a multifunctional protein,regulates cellular function and aging via genetic and epigenetic pathways

Ewing's sarcoma (EWS) is a bone cancer arising predominantly in young children. EWSR1 (Ewing Sarcoma breakpoint region 1/EWS RNA binding protein 1) gene is ubiquitously expressed in most cell types, indicating it has diverse roles in various cellular processes and organ development. Recently, several studies have shown that missense mutations of EWSR1 genes are known to be associated with central nervous system disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Otherwise, EWSR1 plays epigenetic roles in gene expression, RNA processing, and cellular signal transduction. Interestingly, EWSR1 controls micro RNA (miRNA) levels via Drosha, leading to autophagy dysfunction and impaired dermal development. Ewsr1 deficiency also leads to premature senescence of blood cells and gamete cells with a high rate of apoptosis due to the abnormal meiosis. Despite these roles of EWSR1 in various cellular functions, the exact mechanisms are not yet understood. In this context, the current review overviews a large body of evidence and discusses on what EWSR1 genetic mutations are associated with brain diseases and on how EWSR1 modulates cellular function via the epigenetic pathway. This will provide a better understanding of bona fide roles of EWSR1 in aging and its association with brain disorders.     

Genetic requirements and meiotic function of phosphorylation of the yeast axial element protein Red1

The synaptonemal complex (SC) is a meiosis-specific tripartite structure that forms between two homologous chromosomes; it consists of a central region and two parallel lateral elements. Lateral elements also are called axial elements prior to synapsis. In Saccharomyces cerevisiae, Red1, Hop1, and Mek1 are structural components of axial/lateral elements. The red1/mek1/hop1 mutants all exhibit reduced levels of interhomolog recombination and produce no viable spores. Red1 is a phosphoprotein. Several earlier reports proposed that phosphorylated Red1 plays important roles in meiosis, including in signaling meiotic DNA damage or in preventing exit from the pachytene chromosomes. We report here that the phosphorylation of Red1 is carried out in CDC28-dependent and CDC28-independent manners. In contrast to previous results, we found Red1 phosphorylation to be independent of meiotic DNA recombination, the Mec1/Tel1 DNA damage checkpoint kinases, and the Mek1 kinase. To functionally validate the phosphorylation of Red1, we mapped the phosphorylation sites on this protein. A red1(14A) mutant showing no detectable Red1 phosphorylation did not exhibit decreased sporulation efficiency, defects in viable spore production, or defects in meiotic DNA damage checkpoints. Thus, our results suggest that the phosphorylation of Red1 is not essential for its functions in meiosis.     

The yeast 2-micron plasmid Rep2 protein has Rep1-independent partitioning function

Equal partitioning of the multi-copy 2-micron plasmid of the budding yeast Saccharomyces cerevisiae requires association of the plasmid Rep1 and Rep2 proteins with the plasmid STB partitioning locus. Determining how the Rep proteins contribute has been complicated by interactions between the components. Here, each Rep protein was expressed fused to the DNA-binding domain of the bacterial repressor protein LexA in yeast harboring a replication-competent plasmid that had LexA-binding sites but lacked STB. Plasmid transmission to daughter cells was increased only by Rep2 fusion expression. Neither Rep1 nor a functional RSC2 complex (a chromatin remodeler required for 2-micron plasmid partitioning) were needed for the improvement. Deletion analysis showed the carboxy-terminal 65 residues of Rep2 were required and sufficient for this Rep1-independent inheritance. Mutation of a conserved basic motif in this domain impaired Rep1-independent and Rep protein/STB-dependent plasmid partitioning. Our findings suggest Rep2, which requires Rep1 and the RSC2 complex for functional association with STB, directly participates in 2-micron plasmid partitioning by linking the plasmid to a host component that is efficiently partitioned during cell division. Further investigation is needed to reveal the host factor targeted by Rep2 that contributes to the survival of these plasmids in their budding yeast hosts.     

Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p

The nucleolar protein Nep1 and its human homologue were previously shown to be involved in the maturation of 18S rRNA and to interfere directly or indirectly with a methylation reaction. Here, we report that the loss-of-function mutation Δsnr57 and multicopy expression of the ribosomal 40S subunit protein 19 (Rps19p) can partially suppress the Saccharomyces cerevisiae Δnep1 growth defect. SnR57 mediates 2′-O-ribose-methylation of G₁₅₇₀ in the 18S rRNA. By performing a three-hybrid screen, we isolated several short RNA sequences with strong binding affinity to Nep1p. All isolated RNAs shared a six-nucleotide consensus motif C/UUCAAC. Furthermore, one of the isolated RNAs exactly corresponded to nucleotides 1553–1577 of the 18S rRNA, which includes G₁₅₇₀, the site of snR57-dependent 18S rRNA methylation. From protein–protein crosslink data and the cryo-EM map of the S. cerevisiae small ribosomal subunit, we suggest that Rps19p is localized in close vicinity to the Nep1p 18S rRNA binding site. Our results suggest that Nep1p binds adjacent to helix 47 of the 18S rRNA and possibly supports the association of Rps19p to pre-ribosomal particles.     

Properties of an abundant RNA-binding protein in yeast mitochondria

We have previously identified a protein with Mr approximately 40,000 (p40) that binds with high specificity and affinity to the 5'-untranslated leaders of mitochondrial mRNAs in yeast. Here we show that this protein is abundant, comprising about 0.4% of total mitochondrial protein. p40 is present in a cytoplasmic (rho degree) petite mutant that lacks mitochondrial protein synthesis and is therefore nuclear encoded. p40 can be detected by immunological techniques in cell lysates of several different pet mutants, specifically disturbed in the translation of individual mitochondrial mRNAs. It is thus not one of the translation factors defined by any of these mutations. In the case of a pet111 mutant, which is specifically blocked in the translation of COX2 mRNA, extracts still display COX2 mRNA binding activity, indicating that p40 complex formation in vitro is not dependent on the presence of PET111.