Refereed Preprints

Review Commons provides authors with a Refereed Preprint, which includes the authors’ manuscript, reports from a single round of peer review and the authors’ response. These Refereed Preprints are transferred on the author’s behalf to bioRxiv. The most recently-completed Review Commons peer-reviews are listed below, with the most recently posted reviews at the top.

Latest Refereed Preprints

Microbiology | 31 Jul 2020
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Ebola and Marburg virus infection in bats induces a systemic response

Anitha D. Jayaprakash, Adam J. Ronk, Abhishek N. Prasad, Michael F. Covington, Kathryn R. Stein, Toni M. Schwarz, Saboor Hekmaty, Karla A. Fenton, Thomas W Geisbert, Christopher F. Basler, Alexander Bukreyev, Ravi Sachidanandam

The filoviruses Ebola (EBOV) and Marburg (MARV) cause fatal disease in humans and nonhuman primates but are associated with subclinical infections in bats, with Egyptian rousette bat (ERB, Rousettus aegyptiacus) being a natural MARV reservoir. To understand the nature of this resistance, we have analyzed how EBOV and MARV affect the transcriptomes of multiple ERB tissues. We have found that while the primary locus of infection was the liver, gene expression was affected in multiple tissues, suggesting a systemic response. We have identified transcriptional changes that are indicative of inhibition of the complement system, induction of vasodilation, changes in coagulation, modulation of iron regulation, activation of a T cell response, and converting macrophages from the M1 to M2 state. We propose that these events are facets of a systemic anti-inflammatory state that enables effective control of the infection in bats and suggest that dissecting this state can inform how to control a filovirus infection in humans.
Genomics | 21 Jul 2020
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Increased processing of SINE B2 non coding RNAs unveils a novel type of transcriptome de-regulation underlying amyloid beta neuro-pathology

Yubo Cheng, Babita Gollen, Luke Saville, Christopher Isaac, Jogender Mehla, Majid Mohajerani, Athanasios Zovoilis

More than 97% of the mammalian genome is non-protein coding, and repetitive elements account for more than 50% of noncoding space. However, the functional importance of many non-coding RNAs generated by these elements and their connection with pathologic processes remains elusive. We have previously shown that B2 RNAs, a class of non-coding RNAs that belong to the B2 family of SINE repeats, mediate the transcriptional activation of stress response genes (SRGs) upon application of a stimulus. Notably, B2 RNAs bind RNA Polymerase II (RNA Pol II) and suppress SRG transcription during pro-stimulation state. Upon application of a stimulus, B2 RNAs are processed into fragments and degraded, which in turn releases RNA Pol II from suppression and upregulates SRGs. Here, we demonstrate a novel role for B2 RNAs in transcriptome response to amyloid beta toxicity and pathology in the mouse hippocampus. In healthy hippocampi, activation of SRGs is followed by a transient upregulation of pro-apoptotic factors, such as p53 and miRNA-34c, which target SRGs creating a negative feedback loop that facilitates transition to the pro-stimulation state. Using an integrative RNA genomics approach, we show that in mouse hippocampi of an amyloid precursor protein knock-in mouse model and in an in vitro cell culture model of amyloid beta toxicity, this regulatory loop is dysfunctional due to increased levels of B2 RNA processing, constitutively elevated SRG expression and high p53 levels. Evidence indicates that Hsf1, a master regulator of stress response, mediates B2 RNA processing in cells, and is upregulated during amyloid toxicity accelerating the processing of SINE RNAs and SRG hyper-activation. Our study reveals that in mouse, SINE RNAs constitute a novel pathway deregulated in amyloid beta pathology, with potential implications for similar cases in the human brain, such as Alzheimer’s disease (AD). This data attributes a role to SINE RNA processing in a pathological process as well as a new function to Hsf1 that is independent of its transcription factor activity.
Cell Biology | 17 Jul 2020
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Novel features of centriole polarity and cartwheel stacking revealed by cryo-tomography

Sergey Nazarov, Alexandra Bezler, Georgios N Hatzopoulos, Veronika Nemčíková Villímová, Davide Demurtas, Maeva Le Guennec, Paul Guichard, Pierre Gönczy

Centrioles are polarized microtubule-based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers of SAS-6 proteins. A cryo-tomography map of centrioles from the termite flagellate Trichonympha spp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub-tomogram averaging (STA) in T. spp. and Trichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead and A-C-linker. Moreover, we report ∼25 Å resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore, STA of centrioles from the distant flagellate Teranympha mirabilis uncovers similar cartwheel architecture and a distinct filamentous CID. Fitting the CrSAS-6 crystal structure into the flagellate maps and analyzing cartwheels generated in vitro indicates that SAS-6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improved STA maps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms.
Microbiology | 05 Mar 2020
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In clonal cultures, not all cells are equally susceptible to virus infection. Underlying mechanisms of infection variability are poorly understood. Here, we developed image-based single cell measurements to scrutinize the heterogeneity of adenovirus (AdV) infection. AdV delivers, transcribes and replicates a linear double-stranded DNA genome in the nucleus. We measured the abundance of viral transcripts by single-molecule RNA fluorescence in situ hybridization (FISH), and the incoming ethynyl-deoxy-cytidine (EdC)-tagged viral genome by copper(I)-catalyzed azide-alkyne cycloaddition (click) reaction. The early transcripts increased from 2-12 hours, the late ones from 12-23 hours post infection (pi), indicating distinct accumulation kinetics. Surprisingly, the expression of the immediate early transactivator gene E1A only moderately correlated with the number of viral genomes in the cell nucleus, although the incoming viral DNA remained largely intact until 7 hours pi. Genome-to-genome heterogeneity was found at the level of viral transcription, as indicated by colocalization with the large intron containing early region E4 transcripts, uncorrelated to the multiplicity of incoming genomes in the nucleus. In accordance, individual genomes exhibited heterogeneous replication activity, as shown by single-strand DNA-FISH and immunocytochemistry. These results indicate that the variability in viral gene expression and replication are not due to defective genomes but due to host cell heterogeneity. By analyzing the cell cycle state, we found that G1 cells exhibited the highest E1A expression, and significantly increased the correlation between E1A expression and viral genome copy numbers. This combined image-based single molecule procedure is ideally suited to explore the cell-to-cell variability in viral infection, including transcriptional activators and repressors, RNA splicing mechanisms, and the impact of the 3-dimensional nuclear topology on gene regulation.Author SummaryAdenoviruses (AdV) are ubiquitous pathogens in vertebrates. They persist in infected people, and cause unpredictable outbreaks, morbidity and mortality across the globe. Here we report that the common human AdV type C5 (AdV-C5) gives rise to considerable infection variability at the level of single cells in culture, and that a major underlying reason is the cell-to-cell heterogeneity. By combining sensitive single molecule in situ technology for detecting the incoming viral DNA and newly synthesized viral transcripts we show that viral gene expression is heterogeneous between infected human cells, as well as individual genomes. We report a moderate correlation between the number of viral genomes in the nucleus and immediate early E1A transcripts. This correlation is increased in the G1 phase of the cell cycle, where the E1A transcripts were found to be more abundant than in any other cell cycle phase. Our results demonstrate the importance of cell-to-cell variability measurements for understanding transcription and replication in viral infections.
Cell Biology | 08 May 2020
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In mouse oocytes, acentriolar MTOCs functionally replace centrosomes and act as microtubule nucleation sites. Microtubules nucleated from MTOCs initially assemble into an unorganized ball-like structure, which then transforms into a bipolar spindle carrying MTOCs at its poles, a process called spindle bipolarization. In mouse oocytes, spindle bipolarization is promoted by kinetochores but the mechanism by which kinetochore–microtubule attachments contribute to spindle bipolarity remains unclear. This study demonstrates that the stability of kinetochore–microtubule attachment is essential for confining MTOC positions at the spindle poles and for limiting spindle elongation. MTOC sorting is gradual and continues even in the metaphase spindle. When stable kinetochore–microtubule attachments are disrupted, the spindle is unable to restrict MTOCs at its poles and fails to terminate its elongation. Stable kinetochore fibers are directly connected to MTOCs and to the spindle poles, and thus may serve as a measure that defines proper spindle length. These findings reinforce the hypothesis that kinetochores act as scaffolds for acentrosomal spindle bipolarity.
Molecular Biology | 15 Jul 2020
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The lupus autoantigen La is an Xist-binding protein involved in Xist folding and cloud formation

Norbert Ha, Nan Ding, Ru Hong, Rubing Liu, Xavier Roca, Yingyuan Luo, Xiaowei Duan, Xiao Wang, Peiling Ni, Li-Feng Zhang, Lingyi Chen

Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, the lupus autoantigen La (encoding gene Ssb) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female ES cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. La is a ubiquitous and versatile RNA-binding protein with RNA chaperone and RNA helicase activities. Functional dissection of La shows that the RNA chaperone domain and/or the ATP binding motif play critical roles in XCI. In mutant cells, Xist transcripts are unstable and misfolded. These results show that La is critically involved in XCI, possibly as a protein regulating the in-cell structure of Xist.
Molecular Biology | 07 May 2020
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Transcriptional comparison of Testicular Adrenal Rest Tumors with fetal and adult tissues

Mariska A.M. Schröder, Fred C.G.J. Sweep, Antonius E. van Herwaarden, Alan E. Rowan, Darren Korbie, Rod T. Mitchell, Nike M.M.L. Stikkelbroeck, Hedi L. Claahsen – van der Grinten, Paul N. Span

Testicular Adrenal Rest Tumors (TART) are a common complication of unknown origin in patients with Congenital Adrenal Hyperplasia. These benign tumors may derive from cells of adrenal origin or from pluripotent progenitor cells from the fetal adrenogonadal primordium. By comparing the transcriptome of TART with fetal- and adult-testis and adrenal tissues, this study aims to unravel the origin of TART. Targeted transcriptome sequencing was followed by unsupervised clustering-, differential expression-, functional enrichment- and pathway analyses. Immunohistochemistry demonstrated co-expression of adrenal-specific CYP11B1 and testis-specific HSD17B3 in TART, indicating the existence of a distinct TART cell exhibiting both adrenal- and testicular characteristics. Principal component analysis indicated that the TART transcriptome was distinct from either adrenal or testis fetal tissue, making a progenitor-like phenotype of TART unlikely. Rather, TART appears to originate from -or differentiate into-a mature cell type, with both adrenal- and testicular characteristics. The present study, the first to describe the TART transcriptome, expands knowledge about the identity and functional characteristics of TART and identifies clinically targetable pathways associated with fibrosis.
Developmental Biology | 23 Apr 2020
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Vasohibin-1 mediated tubulin detyrosination selectively regulates secondary sprouting and lymphangiogenesis in the zebrafish trunk

Bastos de Oliveira Marta, Meier Katja, Coxam Baptiste, Geudens Ilse, Jung Simone, Szymborska Anna, Gerhardt Holger

Previous studies have shown that Vasohibin-1 (Vash-1) is stimulated by VEGFs in endothelial cells and that its overexpression interferes with angiogenesis in vivo. Recently, Vasohibin-1 was found to mediate tubulin detyrosination, a post-translational modification that is implicated in many cell functions, such as cell division. Here we used the zebrafish embryo to investigate the cellular and subcellular mechanisms of Vash-1 on endothelial microtubules during formation of the trunk vasculature. We show that microtubules within venous-derived secondary sprouts are strongly and selectively detyrosinated in comparison with other endothelial cells, and that this difference is lost upon vash-1 knockdown. Vasohibin-1 depletion in zebrafish specifically affected secondary sprouting from the posterior cardinal vein, increasing both the number of sprouts and endothelial cell divisions. We show that altering secondary sprout numbers and structure upon vash-1 depletion leads to a failure in the development and specification of lymphatic vessels of the zebrafish trunk.Vasohibin-1 mediated detyrosination of endothelial microtubules is selectively required for adequate behaviour of venous secondary sprouting and subsequent formation of functional lymphatics in the zebrafish trunk.
Immunology | 20 Dec 2019
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Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR)

Rui Dong, Kendra A. Libby, Franziska Blaeschke, Alexander Marson, Ronald D. Vale, Xiaolei Su

The chimeric antigen receptor (CAR) directs T cells to target and kill specific cancer cells. Despite the success of CAR T therapy in clinics, the intracellular signaling pathways that lead to CAR T cell activation remain unclear. Using CD19 CAR as a model, we report that, similar to the endogenous T cell receptor (TCR), antigen-engagement triggers the formation of CAR microclusters that transduce downstream signaling. However, CAR microclusters do not coalesce into a stable central supramolecular activation cluster (cSMAC). Moreover, LAT, an essential scaffold protein for TCR signaling, is not required for microcluster formation, immunological synapse formation, and actin remodeling following CAR activation. Meanwhile, CAR T cells still require LAT for the normal production of the cytokine IL-2. Together, these data show that CAR T cells can bypass LAT for a subset of downstream signaling outputs, thus revealing a rewired signaling pathway as compared to native T cells.
Cell Biology | 03 May 2020
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Homology-directed repair involves multiple strand invasion cycles in fission yeast

Amanda J. Vines, Kenneth Cox, Bryan A. Leland, Megan C. King

Homology-directed repair of DNA double-strand breaks (DSBs) can be a highly faithful pathway. Non-crossover repair dominates in mitotically growing cells, likely through a preference for synthesis-dependent strand annealing (SDSA). While genetic studies highlight a key role for the RecQ helicase BLM/Rqh1 (in human and S. pombe cells, respectively) in promoting noncrossover repair, how homology-directed repair mechanism choice is orchestrated in time and space is not well understood. Here, we develop a microscopy-based assay in living fission yeast to determine the dynamics and kinetics of an engineered, site-specific interhomologue repair event. We observe highly efficient homology search and homology-directed repair in this system. Surprisingly, we find that the initial distance between the DSB and the donor sequence does not correlate with the duration of repair. Instead, we observe that repair is likely to involve multiple site-specific and Rad51-dependent co-localization events between the DSB and donor sequence, suggesting that efficient interhomologue repair in fission yeast often involves multiple strand invasion events. By contrast, we find that loss of Rqh1 leads to successful repair through a single strand invasion event, suggesting that multiple strand invasion cycles reflect ongoing SDSA. However, failure to repair is also more likely in rqh1Δ cells, which could reflect increased strand invasion at non-homologous sites. This work has implications for the molecular etiology of Bloom syndrome, caused by mutations in BLM and characterized by aberrant sister chromatid crossovers and inefficient repair.
Molecular Biology | 23 Apr 2020
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ESI mutagenesis: A one-step method for introducing point mutations into bacterial artificial chromosome transgenes

Arnaud Rondelet, Andrei Pozniakovsky, Marit Leuschner, Ina Poser, Andrea Ssykor, Julian Berlitz, Nadine Schmidt, Anthony A Hyman, Alexander W Bird

Bacterial artificial chromosome (BAC)-based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. While homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call ESI (Exogenous/Synthetic Intronization) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI-mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.
Cell Biology | 10 May 2020
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The establishment of branched structures by single cells involves complex cytoskeletal remodelling events. In Drosophila, epithelial tracheal system terminal cells (TCs) and dendritic arborisation neurons are models for these subcellular branching processes. During tracheal embryonic development, the generation of subcellular branches is characterized by extensive remodelling of the microtubule (MT) network and actin cytoskeleton, followed by vesicular transport and membrane dynamics. We have previously shown that centrosomes are key players in the initiation of subcellular lumen formation where they act as microtubule organizing centres (MTOCs). However, not much is known on the events that lead to the growth of these subcellular luminal branches or what makes them progress through a particular trajectory within the cytoplasm of the TC. Here, we have identified that the spectraplakin Short-stop (Shot) promotes the crosstalk between MTs and actin, which leads to the extension and guidance of the subcellular lumen within the TC cytoplasm. Shot is enriched in cells undergoing the initial steps of subcellular branching as a direct response to FGF signalling. An excess of Shot induces ectopic acentrosomal branching points in the embryonic and larval tracheal TC leading to cells with extra subcellular lumina. These data provide the first evidence for a role for spectraplakins in subcellular lumen formation and branching.
Genomics | 17 Apr 2020
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Pervasive enhancer transcription is at the origin of more than half of all long noncoding RNAs in humans. Transcription of enhancer-associated long noncoding RNAs (elncRNA) contribute to their cognate enhancer activity and gene expression regulation in cis. Recently, splicing of elncRNAs was shown to be associated with elevated enhancer activity. However, whether splicing of elncRNA transcripts is a mere consequence of accessibility at highly active enhancers or if elncRNA splicing directly impacts enhancer function, remains unanswered.We analysed genetically driven changes in elncRNA expression, in humans, to address this outstanding question. We showed that splicing related motifs within multi-exonic elncRNAs evolved under selective constraints during human evolution, suggesting the processing of these transcripts is unlikely to have resulted from transcription across spurious splice sites. Using a genome-wide and unbiased approach, we used nucleotide variants as independent genetic factors to directly assess the causal relationship that underpin elncRNA splicing and their cognate enhancer activity. We found that the splicing of most elncRNAs is associated with changes in chromatin signatures at cognate enhancers and target mRNA expression.We conclude that efficient and conserved processing of enhancer-associated elncRNAs contributes to enhancer activity.
Epidemiology | 02 Mar 2020
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Profiles of circulating histidine-rich glycoprotein associate with chronological age and risk of all-cause mortality

Mun-Gwan Hong, Tea Dodig-Crnković, Xu Chen, Kimi Drobin, Woojoo Lee, Yunzhang Wang, Fredrik Edfors, David Kotol, Cecilia Engel Thomas, Ronald Sjöberg, Jacob Odeberg, Anders Hamsten, Angela Silveira, Per Hall, Peter Nilsson, Yudi Pawitan, Sara Hägg, Mathias Uhlén, Nancy L. Pedersen, Patrik K. E. Magnusson, Jochen M. Schwenk

1.Despite recognizing aging as risk factor of human diseases, little is still known about the molecular traits of biological age and mortality risk. To identify age-associated proteins circulating human blood, we screened 156 subjects aged 50-92 years using an exploratory and multiplexed affinity proteomics approach. We corroborated the top age-associated protein profile (adjusted P < 0.001) in eight additional study sets (N = 4,044 individuals), and confirmed a consistent age-associated increase (P = 6.61 × 10-6) by meta-analysis. Applying antibody validation determined circulating histidine-rich glycoprotein (HRG) as the target, and we observed that sequence variants influenced the antibodies ability to bind to the protein. Profiles of circulating HRG were associated to several clinical traits and predicted the risk of mortality during a follow-up period of 8.5 years (IQR = 7.7-9.3 years) after blood sampling (HR = 1.25 per SD; 95% CI = 1.12-1.39; P = 7.41 × 10-5). In conclusion, our affinity proteomics analysis found associations between the molecular traits of circulating HRG with age and all-cause mortality. This suggests that the profiles of multi-purpose protein HRG could serve as an accessible indicator of physiological processes related to aging.
Biochemistry | 23 Jun 2020
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Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

Sara Sajko, Irina Grishkovskaya, Julius Kostan, Melissa Graewert, Kim Setiawan, Linda Trübestein, Korbinian Niedermüller, Charlotte Gehin, Antonio Sponga, Martin Puchinger, Anne-Claude Gavin, Thomas Leonard, Dimitri Svergun, Terry K. Smith, Brooke Morriswood, Kristina Djinovic-Carugo

MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats – Trypanosoma brucei MORN1. Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation.
Cell Biology | 13 Apr 2020
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Mitochondrial dysfunction causes muscle wasting (or atrophy) in many diseases and probably also during aging. The underlying mechanism is unclear. Accumulating evidence suggests that substantial levels of bioenergetic deficiency and oxidative stress are insufficient by themselves to intrinsically cause muscle wasting, raising the possibility that non-bioenergetic factors may contribute to mitochondria-induced muscle wasting. In this report, we show that chronic adaptation to mitochondria-induced proteostatic stress in the cytosol induces muscle wasting. We generated transgenic mice with unbalanced mitochondrial protein loading and import, by a two-fold increase in the expression of the nuclear-encoded mitochondrial carrier protein, Ant1. We found that the ANT1-transgenic mice progressively lose muscle mass. Skeletal muscle is severely atrophic in older mice without affecting the overall lifespan. Mechanistically, Ant1 overloading induces aggresome-like structures and the expression of small heat shock proteins in the cytosol. The data support mitochondrial Precursor Overaccumulation Stress (mPOS), a recently discovered cellular stress mechanism caused by the toxic accumulation of unimported mitochondrial precursors/preproteins. Importantly, the ANT1-transgenic muscles have a drastically remodeled transcriptome that appears to be trying to counteract mPOS, by repressing protein synthesis, and by stimulating proteasomal function, autophagy and lysosomal amplification. These anti-mPOS responses collectively reduce protein content, which is known to decrease myofiber size and muscle mass. Our work therefore revealed that a subtle imbalance between mitochondrial protein load and import is sufficient to induce mPOS in vivo, and that anti-mPOS adaptation is a robust mechanism of muscle wasting. This finding may help improve the understanding of how mitochondria contribute to muscle wasting. It could have direct implications for several human diseases associated with ANT1 overexpression, including Facioscapulohumeral Dystrophy (FSHD).One Sentence SummaryProteostatic adaptations to proteostatic stress in the cytosol caused by unbalanced mitochondrial protein loading and import lead to progressive muscle wasting.
Cell Biology | 14 Apr 2020
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Alpha-satellite RNA transcripts are repressed by centromere-nucleolus associations

Leah Bury, Brittania Moodie, Liliana S. McKay, Karen H. Miga, Iain M. Cheeseman

Centromeres play a fundamental role in chromosome segregation. Although originally thought to be silent chromosomal regions, centromeres are actively transcribed. However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA smFISH foci fluctuate in their levels over the cell cycle and do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Our results demonstrate that alpha-satellite expression occurs through RNA Polymerase II-dependent transcription, but does not require centromere proteins and other cell division components. Instead, our work implicates centromere-nucleolar associations as the major factor regulating alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels, explaining variations in alpha-satellite RNA between cell lines. In addition, alpha-satellite transcript levels increase substantially when the nucleolus is disrupted. Together, our results are inconsistent with a direct, physical role for alpha-satellite transcripts in cell division processes, and instead support a role for ongoing transcription in promoting centromere chromatin dynamics. The control of alpha-satellite transcription by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions.