Stress granule formation helps to mitigate neurodegeneration.

Cellular stress pathways that inhibit translation initiation lead to transient formation of cytoplasmic RNA/protein complexes known as stress granules. Many of the proteins found within stress granules and the dynamics of stress granule formation and dissolution are implicated in neurodegenerative disease. Whether stress granule formation is protective or harmful in neurodegenerative conditions is not known. To address this, we took advantage of the alphavirus protein nsP3, which selectively binds dimers of the central stress granule nucleator protein G3BP (rin in Drosophila) and markedly reduces stress granule formation without directly impacting the protein translational inhibitory pathways that trigger stress granule formation. In Drosophila and rodent neurons, reducing stress granule formation with nsP3 had modest impacts on lifespan even in the setting of serial stress pathway induction. In contrast, reducing stress granule formation in models of ataxia, amyotrophic lateral sclerosis and frontotemporal dementia largely exacerbated disease phenotypes. These data support a model whereby stress granules mitigate, rather than promote, neurodegenerative cascades.

Developing Future Biologists: developmental biology for undergraduates from underserved communities.

Developing Future Biologists (DFB) is an inclusive, trainee-run organization that strives to excite and engage the next generation of biologists, regardless of race, gender or socioeconomic status, in the field of developmental biology. DFB offers a week-long course consisting of active lectures, hands-on laboratory sessions, and professional development opportunities through interactions with scientists from a variety of backgrounds and careers. A major goal of DFB is to propel undergraduate students from underserved communities to pursue biomedical research opportunities and advanced degrees in science. To achieve this goal, we provide DFB participants with continuing access to a diverse network of scientists that students can utilize to secure opportunities and foster success throughout multiple stages of their research careers. Here, we describe the flourishing DFB program at the University of Michigan to encourage other institutions to create their own DFB programs.

Mechanistic convergence across initiation sites for RAN translation in fragile X associated tremor ataxia syndrome.

Repeat associated non-AUG (RAN) translation of CGG repeats in the 5'UTR of FMR1 produces toxic proteins that contribute to fragile X-associated tremor/ataxia syndrome (FXTAS) pathogenesis. The most abundant RAN product, FMRpolyG, initiates predominantly at an ACG upstream of the repeat. Accurate FMRpolyG measurements in FXTAS patients are lacking. We used data-dependent acquisition and parallel reaction monitoring (PRM) mass spectrometry coupled with stable isotope labeled standard peptides to identify signature FMRpolyG fragments in patient samples. Following immunoprecipitation, PRM detected FMRpolyG signature peptides in transfected cells, and FXTAS tissues and cells, but not in controls. We identified two amino-terminal peptides: an ACG-initiated Ac-MEAPLPGGVR and a GUG-initiated Ac-TEAPLPGGVR, as well as evidence for RAN translation initiation within the CGG repeat itself in two reading frames. Initiation at all sites increased following cellular stress, decreased following eIF1 overexpression and was eIF4A and M7G cap-dependent. These data demonstrate that FMRpolyG is quantifiable in human samples and FMR1 RAN translation initiates via similar mechanisms for near-cognate codons and within the repeat through processes dependent on available initiation factors and cellular environment.

Induction of IL19 expression through JNK and cGAS-STING modulates DNA damage-induced cytokine production.

Cytokine production is a critical component of cell-extrinsic responses to DNA damage and cellular senescence. Here, we demonstrated that expression of the gene encoding interleukin-19 (IL-19) was enhanced by DNA damage through pathways mediated by c-Jun amino-terminal kinase (JNK) and cGAS-STING and that IL19 expression was required for the subsequent production of the cytokines IL-1, IL-6, and IL-8. IL19 expression was stimulated by diverse cellular stresses, including inhibition of the DNA replication checkpoint kinase ATR (ataxia telangiectasia and Rad3-related protein), oncogene expression, replicative exhaustion, oxidative stress, and DNA double-strand breaks. Unlike the production of IL-6 and IL-8, IL19 expression was not affected by abrogation of signaling by the IL-1 receptor (IL-1R) or the mitogen-activated protein kinase p38. Instead, the DNA damage­induced production of IL-1, IL-6, and IL-8 was substantially reduced by suppression of IL19 expression. The signaling pathways required to stimulate IL19 expression selectively depended on the type of DNA-damaging agent. Reactive oxygen species and the ASK1-JNK pathway were critical for responses to ionizing radiation (IR), whereas the cGAS-STING pathway stimulated IL19 expression in response to either IR or ATR inhibition. Whereas induction of IL1, IL6, and IL8 by IR depended on IL19 expression, the cGAS-STING­dependent induction of the immune checkpoint gene PDL1 after IR and ATR inhibition was independent of IL19. Together, these results suggest that IL-19 production by diverse pathways forms a distinct cytokine regulatory arm of the response to DNA damage.

Human oncoprotein 5MP suppresses general and repeat-associated non-AUG translation via eIF3 by a common mechanism.

eIF5-mimic protein (5MP) is a translational regulatory protein that binds the small ribosomal subunit and modulates its activity. 5MP is proposed to reprogram non-AUG translation rates for oncogenes in cancer, but its role in controlling non-AUG initiated synthesis of deleterious repeat-peptide products, such as FMRpolyG observed in fragile-X-associated tremor ataxia syndrome (FXTAS), is unknown. Here, we show that 5MP can suppress both general and repeat-associated non-AUG (RAN) translation by a common mechanism in a manner dependent on its interaction with eIF3. Essentially, 5MP displaces eIF5 through the eIF3c subunit within the preinitiation complex (PIC), thereby increasing the accuracy of initiation. In Drosophila, 5MP/Kra represses neuronal toxicity and enhances the lifespan in an FXTAS disease model. These results implicate 5MP in protecting cells from unwanted byproducts of non-AUG translation in neurodegeneration.

Enhanced detection of expanded repeat mRNA foci with hybridization chain reaction.

Transcribed nucleotide repeat expansions form detectable RNA foci in patient cells that contribute to disease pathogenesis. The most widely used method for detecting RNA foci, fluorescence in situ hybridization (FISH), is powerful but can suffer from issues related to signal above background. Here we developed a repeat-specific form of hybridization chain reaction (R-HCR) as an alternative method for detection of repeat RNA foci in two neurodegenerative disorders: C9orf72 associated ALS and frontotemporal dementia (C9 ALS/FTD) and Fragile X-associated tremor/ataxia syndrome. R-HCR to both G4C2 and CGG repeats exhibited comparable specificity but > 40 × sensitivity compared to FISH, with better detection of both nuclear and cytoplasmic foci in human C9 ALS/FTD fibroblasts, patient iPSC derived neurons, and patient brain samples. Using R-HCR, we observed that integrated stress response (ISR) activation significantly increased the number of endogenous G4C2 repeat RNA foci and triggered their selective nuclear accumulation without evidence of stress granule co-localization in patient fibroblasts and patient derived neurons. These data suggest that R-HCR can be a useful tool for tracking the behavior of repeat expansion mRNA in C9 ALS/FTD and other repeat expansion disorders.

Correction to: HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression.

[This corrects the article DOI: 10.1038/celldisc.2016.45.].

Deletion of ULS1 confers damage tolerance in sgs1 mutants through a Top3-dependent D-loop mediated fork restart pathway.

Homologous recombination (HR)-based repair during DNA replication can apparently utilize several partially overlapping repair pathways in response to any given lesion. A key player in HR repair is the Sgs1-Top3-Rmi1 (STR) complex, which is critical for resolving X-shaped recombination intermediates formed following bypass of methyl methanesulfonate (MMS)-induced damage. STR mutants are also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU), but unlike MMS treatment, HU treatment is not accompanied by X-structure accumulation, and it is thus unclear how STR functions in this context. Here we provide evidence that HU-induced fork stalling enlists Top3 prior to recombination intermediate formation. The resistance of sgs1Δ mutants to HU is enhanced by the absence of the putative SUMO (Small Ubiquitin MOdifier)-targeted ubiquitin ligase, Uls1, and we demonstrate that Top3 is required for this enhanced resistance and for coordinated breaks and subsequent d-loop formation at forks stalled at the ribosomal DNA (rDNA) replication fork block (RFB). We also find that HU resistance depends on the catalytic activity of the E3 SUMO ligase, Mms21, and includes a rapid Rad51-dependent restart mechanism that is different from the slow Rad51-independent HR fork restart mechanism operative in sgs1Δ ULS1+ mutants. These data support a model in which repair of HU-induced damage in sgs1Δ mutants involves an error-prone break-induced replication pathway but, in the absence of Uls1, shifts to one that is higher-fidelity and involves the formation of Rad51-dependent d-loops.

hnRNPA2 mediated acetylation reduces telomere length in response to mitochondrial dysfunction.

Telomeres protect against chromosomal damage. Accelerated telomere loss has been associated with premature aging syndromes such as Werner's syndrome and Dyskeratosis Congenita, while, progressive telomere loss activates a DNA damage response leading to chromosomal instability, typically observed in cancer cells and senescent cells. Therefore, identifying mechanisms of telomere length maintenance is critical for understanding human pathologies. In this paper we demonstrate that mitochondrial dysfunction plays a causal role in telomere shortening. Furthermore, hnRNPA2, a mitochondrial stress responsive lysine acetyltransferase (KAT) acetylates telomere histone H4at lysine 8 of (H4K8) and this acetylation is associated with telomere attrition. Cells containing dysfunctional mitochondria have higher telomere H4K8 acetylation and shorter telomeres independent of cell proliferation rates. Ectopic expression of KAT mutant hnRNPA2 rescued telomere length possibly due to impaired H4K8 acetylation coupled with inability to activate telomerase expression. The phenotypic outcome of telomere shortening in immortalized cells included chromosomal instability (end-fusions) and telomerase activation, typical of an oncogenic transformation; while in non-telomerase expressing fibroblasts, mitochondrial dysfunction induced-telomere attrition resulted in senescence. Our findings provide a mechanistic association between dysfunctional mitochondria and telomere loss and therefore describe a novel epigenetic signal for telomere length maintenance.

Repeat-associated non-AUG (RAN) translation and other molecular mechanisms in Fragile X Tremor Ataxia Syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset inherited neurodegenerative disorder characterized by progressive intention tremor, gait ataxia and dementia associated with mild brain atrophy. The cause of FXTAS is a premutation expansion, of 55 to 200 CGG repeats localized within the 5'UTR of FMR1. These repeats are transcribed in the sense and antisense directions into mutants RNAs, which have increased expression in FXTAS. Furthermore, CGG sense and CCG antisense expanded repeats are translated into novel proteins despite their localization in putatively non-coding regions of the transcript. Here we focus on two proposed disease mechanisms for FXTAS: 1) RNA gain-of-function, whereby the mutant RNAs bind specific proteins and preclude their normal functions, and 2) repeat-associated non-AUG (RAN) translation, whereby translation through the CGG or CCG repeats leads to the production of toxic homopolypeptides, which in turn interfere with a variety of cellular functions. Here, we analyze the data generated to date on both of these potential molecular mechanisms and lay out a path forward for determining which factors drive FXTAS pathogenicity.

RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response.

Repeat-associated non-AUG (RAN) translation allows for unconventional initiation at disease-causing repeat expansions. As RAN translation contributes to pathogenesis in multiple neurodegenerative disorders, determining its mechanistic underpinnings may inform therapeutic development. Here we analyze RAN translation at G4C2 repeat expansions that cause C9orf72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9RAN) and at CGG repeats that cause fragile X-associated tremor/ataxia syndrome. We find that C9RAN translation initiates through a cap- and eIF4A-dependent mechanism that utilizes a CUG start codon. C9RAN and CGG RAN are both selectively enhanced by integrated stress response (ISR) activation. ISR-enhanced RAN translation requires an eIF2α phosphorylation-dependent alteration in start codon fidelity. In parallel, both CGG and G4C2 repeats trigger phosphorylated-eIF2α-dependent stress granule formation and global translational suppression. These findings support a model whereby repeat expansions elicit cellular stress conditions that favor RAN translation of toxic proteins, creating a potential feed-forward loop that contributes to neurodegeneration.

HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression.

Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies.

Enhancing a Wnt-Telomere Feedback Loop Restores Intestinal Stem Cell Function in a Human Organotypic Model of Dyskeratosis Congenita.

Patients with dyskeratosis congenita (DC) suffer from stem cell failure in highly proliferative tissues, including the intestinal epithelium. Few therapeutic options exist for this disorder, and patients are treated primarily with bone marrow transplantation to restore hematopoietic function. Here, we generate isogenic DC patient and disease allele-corrected intestinal tissue using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene correction in induced pluripotent stem cells and directed differentiation. We show that DC tissue has suboptimal Wnt pathway activity causing intestinal stem cell failure and that enhanced expression of the telomere-capping protein TRF2, a Wnt target gene, can alleviate DC phenotypes. Treatment with the clinically relevant Wnt agonists LiCl or CHIR99021 restored TRF2 expression and reversed gastrointestinal DC phenotypes, including organoid formation in vitro, and maturation of intestinal tissue and xenografted organoids in vivo. Thus, the isogenic DC cell model provides a platform for therapeutic discovery and identifies Wnt modulation as a potential strategy for treatment of DC patients.

Resolution by unassisted Top3 points to template switch recombination intermediates during DNA replication.

The evolutionarily conserved Sgs1/Top3/Rmi1 (STR) complex plays vital roles in DNA replication and repair. One crucial activity of the complex is dissolution of toxic X-shaped recombination intermediates that accumulate during replication of damaged DNA. However, despite several years of study the nature of these X-shaped molecules remains debated. Here we use genetic approaches and two-dimensional gel electrophoresis of genomic DNA to show that Top3, unassisted by Sgs1 and Rmi1, has modest capacities to provide resistance to MMS and to resolve recombination-dependent X-shaped molecules. The X-shaped molecules have structural properties consistent with hemicatenane-related template switch recombination intermediates (Rec-Xs) but not Holliday junction (HJ) intermediates. Consistent with these findings, we demonstrate that purified Top3 can resolve a synthetic Rec-X but not a synthetic double HJ in vitro. We also find that unassisted Top3 does not affect crossing over during double strand break repair, which is known to involve double HJ intermediates, confirming that unassisted Top3 activities are restricted to substrates that are distinct from HJs. These data help illuminate the nature of the X-shaped molecules that accumulate during replication of damaged DNA templates, and also clarify the roles played by Top3 and the STR complex as a whole during the resolution of replication-associated recombination intermediates.