A Local Role for the Small Ribosomal Subunit Primary Binder rpS5 in Final 18S rRNA Processing in Yeast

In vivo depletion of the yeast small ribosomal subunit (SSU) protein S5 (rpS5) leads to nuclear degradation of nascent SSUs and to a perturbed global assembly state of the SSU head domain. Here, we report that rpS5 plays an additional local role at the head/platform interface in efficient SSU maturation. We find that yeast small ribosomal subunits which incorporated an rpS5 variant lacking the seven C-terminal amino acids have a largely assembled head domain and are exported to the cytoplasm. On the other hand, 3′ processing of 18S rRNA precursors is inhibited in these ribosomal particles, although they associate with the putative endonuclease Nob1p and other late acting 40S biogenesis factors. We suggest that the SSU head component rpS5 and platform components as rpS14 are crucial constituents of a highly defined spatial arrangement in the head – platform interface of nascent SSUs, which is required for efficient processing of the therein predicted SSU rRNA 3′ end. Positioning of rpS5 in nascent SSUs, including its relative orientation towards platform components in the head-platform cleft, will depend on the general assembly and folding state of the head domain. Therefore, the suggested model can explain 18S precursor rRNA 3′ processing phenotypes observed in many eukaryotic SSU head assembly mutants

rRNA Maturation in Yeast Cells Depleted of Large Ribosomal Subunit Proteins

The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein – rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5′ end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events

PolyQ length-dependent metabolic alterations and DNA damage drive human astrocyte dysfunction in Huntington's disease

Huntington's Disease (HD) is a neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the Huntingtin gene. Astrocyte dysfunction is known to contribute to HD pathology, however our understanding of the molecular pathways involved is limited. Transcriptomic analysis of patient-derived PSC (pluripotent stem cells) astrocyte lines revealed that astrocytes with similar polyQ lengths shared a large number of differentially expressed genes (DEGs). Notably, weighted correlation network analysis (WGCNA) modules from iPSC derived astrocytes showed significant overlap with WGCNA modules from two post-mortem HD cohorts. Further experiments revealed two key elements of astrocyte dysfunction. Firstly, expression of genes linked to astrocyte reactivity, as well as metabolic changes were polyQ length-dependent. Hypermetabolism was observed in shorter polyQ length astrocytes compared to controls, whereas metabolic activity and release of metabolites were significantly reduced in astrocytes with increasing polyQ lengths. Secondly, all HD astrocytes showed increased DNA damage, DNA damage response and upregulation of mismatch repair genes and proteins. Together our study shows for the first time polyQ-dependent phenotypes and functional changes in HD astrocytes providing evidence that increased DNA damage and DNA damage response could contribute to HD astrocyte dysfunction

Generalization of Conditioned Contextual Anxiety and the Modulatory Effects of Anxiety Sensitivity

Anxiety patients overgeneralize fear responses, possibly because they cannot distinguish between cues never been associated with a threat (i.e., safe) and threat-associated cues. However, as contexts and not cues are discussed as the relevant triggers for prolonged anxiety responses characterizing many anxiety disorders, we speculated that it is rather overgeneralization of contextual anxiety, which constitutes a risk factor for anxiety disorders. To this end, we investigated generalization of conditioned contextual anxiety and explored modulatory effects of anxiety sensitivity, a risk factor for anxiety disorders. Fifty-five participants underwent context conditioning in a virtual reality paradigm. On Day 1 (acquisition), participants received unpredictable mildly painful electric stimuli (unconditioned stimulus, US) in one virtual office (anxiety context, CTX+), but never in a

Abnormal molecular signatures of inflammation, energy metabolism, and vesicle biology in human Huntington disease peripheral tissues

BACKGROUND: A major challenge in neurodegenerative diseases concerns identifying biological disease signatures that track with disease progression or respond to an intervention. Several clinical trials in Huntington disease (HD), an inherited, progressive neurodegenerative disease, are currently ongoing. Therefore, we examine whether peripheral tissues can serve as a source of readily accessible biological signatures at the RNA and protein level in HD patients. RESULTS: We generate large, high-quality human datasets from skeletal muscle, skin and adipose tissue to probe molecular changes in human premanifest and early manifest HD patients—those most likely involved in clinical trials. The analysis of the transcriptomics and proteomics data shows robust, stage-dependent dysregulation. Gene ontology analysis confirms the involvement of inflammation and energy metabolism in peripheral HD pathogenesis. Furthermore, we observe changes in the homeostasis of extracellular vesicles, where we find consistent changes of genes and proteins involved in this process. In-depth single nucleotide polymorphism data across the HTT gene are derived from the generated primary cell lines. CONCLUSIONS: Our ‘omics data document the involvement of inflammation, energy metabolism, and extracellular vesicle homeostasis. This demonstrates the potential to identify biological signatures from peripheral tissues in HD suitable as biomarkers in clinical trials. The generated data, complemented by the primary cell lines established from peripheral tissues, and a large panel of iPSC lines that can serve as human models of HD are a valuable and unique resource to advance the current understanding of molecular mechanisms driving HD pathogenesis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13059-022-02752-5

池田婚姻願（宮内大臣宛様式）

The heat shock response (HSR) is a mechanism to cope with proteotoxic stress by inducing the expression of molecular chaperones and other heat shock response genes. The HSR is evolutionarily well conserved and has been widely studied in bacteria, cell lines and lower eukaryotic model organisms. However, mechanistic insights into the HSR in higher eukaryotes, in particular in mammals, are limited. We have developed an in vivo heat shock protocol to analyze the HSR in mice and dissected heat shock factor 1 (HSF1)-dependent and-independent pathways. Whilst the induction of proteostasis-related genes was dependent on HSF1, the regulation of circadian function related genes, indicating that the circadian clock oscillators have been reset, was independent of its presence. Furthermore, we demonstrate that the in vivo HSR is impaired in mouse models of Huntington's disease but we were unable to corroborate the general repression of transcription that follows a heat shock in lower eukaryotes

Characterization of r-protein variants in Saccharomyces cerevisiae

Single ribosomal proteins are required for specific steps in eukaryotic ribosome biogenesis. Consequently, depletion of a certain r-protein leads to a block or delay in pre-rRNA maturation and/or transport of precursor subunits. However, the exact molecular functions of the r-proteins in these processes are still obscure. To accurately investigate the molecular functions and to determine possible multiple roles, variant r-proteins with partial functionality were created and their impact on ribosome biogenesis was analyzed. One set of variant r-proteins was created based on the conservation of r-proteins between the evolutionary kingdoms. In this approach, archaeal r-proteins were expressed in yeast and assayed for conserved functions. The functional characterization of archaeal ribosomal proteins showed the ability of many of them to assemble in vivo into eukaryotic pre-ribosomes. This suggests that r-protein – rRNA interactions are widely conserved between Archaea and Eukarya. Interestingly, incorporation of two archaeal r-proteins into nascent ribosomal subunits promoted their subsequent nuclear export. Apparently, the role of the homologous eukaryotic r-protein in nucleo-cytoplasmic transport is based on evolutionary conserved features and not due to a gain of function in the course of evolution. In an alternative approach, variant r-proteins were created based on current atomic structure models of eukaryotic small subunits, possibly giving insights into structure-function correlation of certain r-proteins. Thorough analysis of rpS5, the primary binder of the head domain revealed a dual role in ribosome biogenesis: A first one in the global organization of the SSU head domain. And a second one in establishment of a highly defined spatial arrangement in the head-platform interface of nascent SSUs that is required for efficient processing of the 18S rRNA 3'-end. This interface most probably senses the overall maturation/assembly state of the head domain of pre-SSUs. Thereby, many phenotypes caused by depletion of other head domain r-proteins can be explained. Furthermore, functional characterization of several variants of rpS2, rpS14 and rpS20 indicated that final cytoplasmic 3'-end processing of eukaryotic 18S rRNA not only depends on a certain protein composition of small ribosomal subunit precursors, but also on their exact conformational state. RNA co-immunoprecipitation experiments and mass spectrometric analyses showed that the sole assembly of the putative nuclease, mediating the final maturation step of SSU precursors, is not sufficient to trigger removal of pre -rRNA sequences. The occurrence of this processing step is therefore consequence of a highly defined assembly and folding state of nascent small ribosomal subunits and might directly correlate with their capability to function in translation

A common gene expression signature in Huntington's disease patient brain regions

BACKGROUND: Gene expression data provide invaluable insights into disease mechanisms. In Huntington’s disease (HD), a neurodegenerative disease caused by a tri-nucleotide repeat expansion in the huntingtin gene, extensive transcriptional dysregulation has been reported. Conventional dysregulation analysis has shown that e.g. in the caudate nucleus of the post mortem HD brain the gene expression level of about a third of all genes was altered. Owing to this large number of dysregulated genes, the underlying relevance of expression changes is often lost in huge gene lists that are difficult to comprehend. METHODS: To alleviate this problem, we employed weighted correlation network analysis to archival gene expression datasets of HD post mortem brain regions. RESULTS: We were able to uncover previously unidentified transcription dysregulation in the HD cerebellum that contained a gene expression signature in common with the caudate nucleus and the BA4 region of the frontal cortex. Furthermore, we found that yet unassociated pathways, e.g. global mRNA processing, were dysregulated in HD. We provide evidence to show that, contrary to previous findings, mutant huntingtin is sufficient to induce a subset of stress response genes in the cerebellum and frontal cortex BA4 region. The comparison of HD with other neurodegenerative disorders showed that the immune system, in particular the complement system, is generally activated. We also demonstrate that HD mouse models mimic some aspects of the disease very well, while others, e.g. the activation of the immune system are inadequately reflected. CONCLUSION: Our analysis provides novel insights into the molecular pathogenesis in HD and identifies genes and pathways as potential therapeutic targets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12920-014-0060-2) contains supplementary material, which is available to authorized users

Contesting the dogma of an age-related heat shock response impairment:implications for cardiac-specific age-related disorders

Ageing is associated with the reduced performance of physiological processes and has been proposed as a major risk factor for disease. An age-related decline in stress response pathways has been widely documented in lower organisms. In particular, the heat shock response (HSR) becomes severely compromised with age in Caenorhabditis elegans. However, a comprehensive analysis of the consequences of ageing on the HSR in higher organisms has not been documented. We used both HS and inhibition of HSP90 to induce the HSR in wild-type mice at 3 and 22 months of age to investigate the extent to which different brain regions, and peripheral tissues can sustain HSF1 activity and HS protein (HSP) expression with age. Using chromatin immunoprecipitation, quantitative reverse transcription polymerase chain reaction, western blotting and enzyme linked immunosorbent assay (ELISA), we were unable to detect a difference in the level or kinetics of HSP expression between young and old mice in all brain regions. In contrast, we did observe an age-related reduction in chaperone levels and HSR-related proteins in the heart. This could result in a decrease in the protein folding capacity of old hearts with implications for age-related cardiac disorder