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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

Ectopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors such as RAD50. We show in yeast and human cells that R-loops accumulate during RAD50 depletion. In human cancer cell models, we find that RAD50 and its partners in the MRE11-RAD50-NBS1 complex regulate R-loop-associated DNA damage and replication stress. We show that a non-nucleolytic function of MRE11 is important for R-loop suppression via activation of PCNA-ubiquitination by RAD18 and recruiting anti-R-loop helicases in the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms of transcription-replication conflicts.

cell biology↗

bioRxiv2018

Transient activation of the UPRER is an essential step in the acquisition of pluripotency during reprogramming

Somatic cells can be reprogrammed into pluripotent stem cells by the forced expression of the OCT4, SOX2, KLF4 and c-MYC transcription factors. This process requires the reshaping of not only epigenetic landscapes, but the global remodeling of cell identity, structure, and function including such basic processes of metabolism and organelle form and function. Cellular reprogramming is a stochastic process with only a marginally measureable fraction of cells successfully crossing these, and many other, cellular epitomes to acquire the fully pluripotent state. We hypothesize that this variation is due, in part, by variable regulation of the proteostasis network and its influence upon the protein folding environment within cells and their organelles upon the remodeling process. We find that the endoplasmic reticulum unfolded protein response (UPRER), the heat-shock response (HSR) and the mitochondrial unfolded protein response (UPRmt), which monitor and ensure the quality of the proteome of, respectively, the ER, the cytosol and the mitochondria during stress, are activated during cellular reprogramming. Particularly, we find that the UPRER is essential for reprograming, and ectopic, transient activation of the UPRER, either genetically or pharmacologically, enhances the success of cells to reach a pluripotent state. Finally, and most revealing, we find that stochastic activation of the UPRER can predict the reprogramming efficiency of naive cells. The results of these experiments indicate that the low efficiency and stochasticity of cellular reprogramming is partly the result of the inability to initiate a proper ER stress response for remodeling of the ER and its proteome during the reprogramming process. The results reported here display only one aspect of the proteostasis network and suggest that proper regulation of many more components of this network might be essential to acquire the pluripotent state.

cell biology↗

bioRxiv2018

Dynamic constriction and fission of ER membranes by reticulon

The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been widely associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. We found that the ER network has an embedded fragmentation mechanism based upon the ability of reticulons to produce fission of elongating network branches. In Drosophila, fission is counterbalanced by atlastin-driven fusion, with their imbalance leading to ER fragmentation. Live imaging of ER network dynamics upon ectopic expression of Drosophila reticulon linked fission to augmented membrane friction. Consistently, in vitro analysis revealed that purified reticulon produced velocity-dependent constriction and fission of lipid nanotubes pulled from a flat reservoir membrane. Fission occurred at elongation rates and pulling force ranges intrinsic to the ER network, thus suggesting a novel principle of organelle morphology regulation where the dynamic balance between fusion and fission is governed by membrane motility.

cell biology↗

bioRxiv2018

Importin-β targets HURP to kinetochore-fibers in coordination with Ran-GTP in human mitotic cells

During mitosis, a bipolar spindle is assembled around chromosomes to efficiently capture chromosomes. Previous work proposed that a chromosome-derived Ran-GTP gradient promotes spindle assembly around chromosomes by liberating spindle assembly factors (SAFs) from inhibitory importins. However, Rans dual functions in interphase nucleocytoplasmic transport and mitotic spindle assembly have made it difficult to assess its mitotic roles in somatic cells. Here, using auxin-inducible degron technology in human cells, we developed acute mitotic degradation assays to dissect Rans mitotic roles systematically and separately from its interphase function. In contrast to the prevailing model, we found that the Ran pathway is not essential for spindle assembly activities that occur at sites spatially separated from chromosomes, including activating NuMA for spindle pole focusing or for targeting TPX2. In contrast, Ran-GTP is required to localize HURP and HSET specifically at chromosome-proximal regions. We demonstrated that Ran-GTP and importin-{beta} coordinately promote HURPs dynamic microtubule binding-dissociation cycle near chromosomes, which results in stable kinetochore-fiber formation. Intriguingly, this pathway acts to establish proper spindle length preferentially during prometaphase, rather than metaphase. Together, we propose that the Ran pathway is required to activate SAFs specifically near chromosomes, but not generally during human mitotic spindle assembly. Ran-dependent spindle assembly is likely coupled with parallel pathways to activate SAFs, including NuMA, for spindle pole focusing away from chromosomes. HighlightsO_LIUsing auxin-inducible degron technology, we developed mitotic degradation assays for the Ran pathway in human cells. C_LIO_LIThe Ran pathway is non-essential to activate NuMA for spindle pole focusing. C_LIO_LIThe Ran pathway dynamically polarizes HURP and defines mitotic spindle length preferentially during prometaphase. C_LIO_LIRan-GTP is required to activate SAFs specifically near chromosomes, but not generally, in human mitotic cells. C_LI

cell biology↗

bioRxiv2018

TCF7L1 and TCF7 differentially regulate specific mouse ES cell genes in response to GSK-3 inhibition

The genome-wide chromatin occupancy of the TCF/LEF factors and its modulation by Wnt pathway activation remain poorly defined. Here, we describe mouse ES cell (mESC) lines expressing a single copy knock-in of the 3xFLAG epitope at the N-terminus of TCF7L1 and TCF7, the two most-highly expressed TCF/LEF factors in mESCs. TCF7L1 protein levels, detected by immunoblotting with a FLAG antibody, were much higher than TCF7 in mESCs maintained in standard serum- and LIF-supplemented medium, even in the presence of the GSK-3 inhibitor, CHIR99021 (CHIR). We used FLAG antibody-mediated ChIP-seq to determine TCF7 and TCF7L1 chromatin occupancy in mESCs cultured in standard medium with or without CHIR for 14 hours. TCF7 and TCF7L1 displayed very few overlapping ChIP peaks across the genome, with TCF7L1 binding significantly more genes than TCF7 in both culture conditions. Despite a reduction in total TCF7L1 protein after CHIR treatment, the TCF7L1 ChIP peak profiles were not uniformly attenuated. Our data demonstrate that TCF7L1 chromatin occupancy upon short-term CHIR treatment is modulated in a target-specific manner. Our findings also suggest that Wnt target genes in mESCs are not regulated by TCF/LEF switching, and TCF7L1, although often called a constitutive repressor, may serve as a transcriptional activator of certain target genes in CHIR-treated mESCs.\n\nHighlightsO_LIChIP and cytometry data suggest that TCF7L1 does not directly regulate mESC Nanog expression.\nC_LIO_LITCF7L1 remains associated with {beta}-catenin in the presence of CHIR99021.\nC_LIO_LITCF7 and TCF7L1 display different chromatin occupancies in mESCs.\nC_LIO_LITCF7L1 binding at specific genomic sites is variably altered by CHIR99021.\nC_LI

cell biology↗

bioRxiv2018

CRISPR/Cas12a-assisted PCR tagging of mammalian genes

Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g. GFP), a Cas12a CRISPR RNA for cleavage of the target locus and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artefacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein tagged genes

cell biology↗

bioRxiv2018

The deSUMOylase SENP2 coordinates homologous recombination and non-homologous end joining by independent mechanisms.

SUMOylation in the DNA double-strand break (DSB) response regulates recruitment, activity and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and non-homologous enjoining (NHEJ) through the investigation of the deSUMOylase SENP2. We find regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast we show HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 foci retention and increases NHEJ and radioresistance. Collectively our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.

cell biology↗

bioRxiv2018

Rac-dependent signaling from keratinocytes promotes differentiation of intradermal white adipocytes

Rac signaling affects numerous downstream targets; however, few studies have established in vivo levels. We generated mice with a single knockout (KO) of Rac1 (Keratin5 (K5)-Cre;Rac1flox/flox, Rac1-KO) and double KO of Rac1 and Rac3 (K5-Cre;Rac1flox/flox;Rac3-/-, Rac1/Rac3-DKO) in keratinocytes. Strikingly, Rac1-KO mice exhibited thinner dermal white adipose tissue, which was considerably further reduced in Rac1/Rac3-DKO mice. DNA microarray using primary keratinocytes from Rac1/Rac3-DKO mice exhibited decreased mRNA levels of Bmp2, Bmp5, Fgf20, Fgf21, Fgfbp1, and Pdgf. Combinational treatment with BMP2 and FGF21 or BMP2 and FGF20 in culture medium, but not individual purified recombinant proteins, could differentiate 3T3-L1 fibroblasts into adipocytes, as could culture media obtained from primary keratinocytes. Conversely, addition of anti-BMP2 or anti-FGF21 antibodies into the culture medium inhibited fibroblast differentiation. Furthermore, combinational treatment with BMP2 and FGF21 promoted adipocyte differentiation only of rat primary white, but not brown, adipocyte precursors. Notably, brown adipogenesis by FGF21 was inhibited by BMP2. Thus, we proposed novel paracrine pathways from keratinocytes to intradermal pre-adipocytes, which function as Rac-dependent modulators of white adipogenesis, but also brown adipogenesis.

cell biology↗

bioRxiv2018

Trypanosoma brucei Pex13.2 is an accessory peroxin that functions in the import of PTS2 proteins and localizes to subdomains of the glycosome

Kinetoplastid parasites including Trypanosoma brucei, Trypanosoma cruzi and Leishmania harbor unique organelles known as glycosomes, which are evolutionarily related to peroxisomes. Glycosome/peroxisome biogenesis is mediated by proteins called peroxins that facilitate organelle formation, proliferation and degradation, and import of proteins housed therein. Import of matrix proteins occurs via one of two pathways that are dictated by their peroxisome targeting sequence (PTS). In PTS1 import, a C-terminal tripeptide sequence, most commonly SKL, is recognized by the soluble receptor Pex5. In PTS2 import, a less conserved N-terminal sequence is recognized by Pex7. The soluble receptors deliver their cargo to the import channel consisting minimally of Pex13 and Pex14. While much of the import process is conserved, kinetoplastids are the only organisms to have two Pex13s, TbPex13.1 and TbPex13.2. In previous studies, GFP-tagged TbPex13.1 localized to glycosomes and silencing either protein in the stage of the parasite that lives in the mammalian bloodstream impaired glycosome protein import and slowed parasite growth. While these findings suggest Pex13s are involved in protein import, the mechanisms by which they function are unknown and it is unclear why kinetoplastids would require two Pex13s. In this work, we demonstrate that TbPex13.2 is associated with the glycosome membrane with its N-terminus facing the cytoplasm. Super-resolution microscopy reveals that TbPex13.2 localizes to a few (1-3) foci per glycosome and import of PTS2 proteins was disrupted in TbPex13.2-deficient cells suggesting it may be an accessory factor for PTS2 import.

cell biology↗

bioRxiv2018

Elevation of intracellular levels of nitric oxide in SHR attenuates hyperproliferation of vascular smooth muscle cells through the inhibition of AT1 receptor expression and c-Src/growth factor receptor signaling pathways

We previously showed that decreased levels of intracellular nitric oxide (NO) contribute to the hyperproliferation of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR). The present study investigates if elevation of intracellular levels of NO by in vivo treatment of SHR with NO donor, sodium nitroprusside (SNP) that was shown to attenuate hypertension could attenuate the hyperproliferation of VSMC and identify the molecular mechanisms. Intraperitoneal injection of SNP (0.5 mg/kg BW) into 8-week-old SHR and WKY rats twice a week for two weeks increased significantly the intracellular levels of NO in aortic VSMC and resulted in the attenuation of hyperproliferation of VSMC from SHR to control levels. The antiproliferative effect of SNP was associated with the restoration of the overexpression of cell cycle proteins, cyclins D1, E, Cdk2, Cdk4, phosphorylated pRB and decreased expression of Cdk inhibitors p21Cip1 and p27Kip1 towards control levels. In addition, SNP treatment also attenuated the overexpression of angiotensin II receptor type 1 (AT1) receptor, phosphorylation of c-Src, EGF-R, PDGF-R, IGF-IR and ERK1/2 in VSMC from SHR to control levels. These results suggest that the augmentation of intracellular levels of NO elicits antiproliferative effect that may be mediated through its ability to inhibit the enhanced expression of AT1 receptor, activation of c- Src, growth factor receptors and MAP kinase signaling and overexpression of cell cycle proteins.

cell biology↗

bioRxiv2018

Effect of continuous compressive force on the expression of RANKL, OPG, and VEGF in MC3T3-E1 and MLO-Y4 cells

Osteocytes, known to have mechano-sensory functions, influence the regulation of bone remodeling. However, the mechanism by which osteocytes regulate bone metabolism when mechanical forces are being applied is still unclear. Osteoclastogenesis is mainly regulated by receptor activator of nuclear factor kappa-B ligand (RANKL); the protein osteoprotegerin (OPG) and angiogenesis also play important roles in osteogenesis. RANKL, OPG, and vascular endothelial growth factor (VEGF) are thought to be key factors for bone metabolism. In this study, we examined the effect of a continuous compressive force (CF) on the expression of RANKL, OPG, and VEGF in osteoblastic murine osteocytes (MLO-Y4) and osteoblastic (MC3T3-E1) cells. Gene and protein expression levels of RANKL, OPG, and VEGF in MLO-Y4 and MC3T3-E1 cells were quantitatively determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). Both cell types were also subjected to a CF of 1.0 g/cm2 for 1, 3, 6, and 12 hours. Furthermore, the effect of a stretch-activated (S-A) channel was examined by gadolinium (Gd3+) administration. The ratio of gene and protein expressions of RANKL, VEGF, and RANKL/OPG in MLO-Y4 cells were significantly higher than in MC3T3-E1 cells, while the expression of OPG was significantly lower. After CF application, both cell types showed significant increases in RANKL and VEGF expression as well as the RANKL/OPG ratio. Additionally, the upregulated gene and protein levels of these factors were reduced by Gd3+ administration.\n\nThese findings suggest that osteocytes play more important roles in bone metabolism and angiogenesis than osteoblasts. Osteocytes regulate the expression of RANKL, OPG, and VEGF via the S-A channel through the response to mechanical stress.

cell biology↗

bioRxiv2018

Title: Human Lung Epithelial Cells Divide >200 Population Doublings without Engaging a Telomere Maintenance Mechanism

The \"Hayflick limit\" is a \"mitotic clock\" and primary cells have a finite lifespan that correlates with telomere length. However, introduction of the telomerase catalytic protein component (TERT) is insufficient to immortalize most, but not all, human cell types under typical cell culture conditions. Originally, telomerase activity was only detected in cancer cells but is now recognized as being detectable in transit amplifying cells in tissues undergoing regeneration or in extreme conditions of wound repair. Here we report that in vitro low stress culture conditions allow normal human lung basal epithelial cells to grow for over 200 population doublings without engaging any telomere maintenance mechanism. This suggests that most reported instances of telomere-based replicative senescence are due to cell culture stress-induced premature senescence.\n\nOne Sentence SummaryHuman lung cells growing in reduced stress conditions can divide well beyond the Hayflick limit.

cell biology↗

bioRxiv2018

Neutrophil Elastase Activates Macrophage MMPs, Promotes Cell Adhesion And Cytokine Production Via Integrin-Src Kinases Pathway

There are a number of diseases characterized by the presence of neutrophil elastase (NE) activity in tissues including cystic fibrosis and alpha-1-antitrypsin deficiency induced lung destruction. It is generally accepted that NE actively contributes to this pathological process, but the precise mechanisms has yet to be determined. We hypothesized that NE activates the macrophages (M{square}) pro-inflammatory program. We demonstrate that following NE exposure, monocyte-derived M{square} release proteolytic activity composed of several matrix metalloproteinases (MMPs) which could contribute to extracellular matrix (ECM) degradation. NE upregulates expression of M{square} derived pro-inflammatory cytokines including TNF, IL-1{beta}, and IL-8. Thus, NE-activated M{square} can contribute to tissue destruction through the proteolytic activity of metalloproteinases and by supporting chronic inflammation through expression of pro-inflammatory cytokines. We also demonstrate that NE increases M{square} adhesion that is attenuated by antibodies specific to integrin subunits. We show that the effects of NE on M{square} can be mediated through an activation of integrin pathways. In support of integrin involvement, we demonstrate that NE activates the Src kinase family, a hallmark of integrin signaling activation. Moreover, pretreatment of macrophages with a specific Src kinase inhibitor, PP2, completely prevents NE-induced inflammatory cytokine production. Taken together these findings indicate that NE has effect on lung destruction that extends beyond direct proteolytic degradation of matrix proteins.

cell biology↗

bioRxiv2018

A genetically encoded probe for imaging HA-tagged protein translation, localization, and dynamics in living cells and animals

To expand the toolbox of imaging in living cells, we have engineered a new single chain variable fragment (scFv) that binds the classic linear HA epitope with high affinity and specificity in vivo. The resulting probe, which we call the HA frankenbody, is capable of lighting up in multiple colors HA-tagged nuclear, cytoplasmic, and membrane proteins in diverse living cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments, which we demonstrate by tracking single mRNA translation dynamics in living U2OS cells and neurons. In combination with the SunTag, we track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful new tool for imaging protein dynamics in vivo.\n\nOne-sentence summaryA genetically encodable intracellular single-chain variable fragment that selectively binds the HA epitope (YPYDVPDYA) with high affinity in living cells and organisms can be used to quantify HA-tagged protein translation, localization, and dynamics.

cell biology↗

bioRxiv2018

Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cells Ex Vivo

Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our novel culture system provides a means to evaluate properties of steady state HSCs and test effects of defined factors in vitro under near-physiological conditions.

cell biology↗

bioRxiv2018

Effects of 5-aza-2 -deoxycytidine on human osteoarthritic chondrocytes

BackgroundGiven regenerative therapies, the utilization of primary human cells is desired and requested in the development of in vitro systems and disease models. After a few passages in vitro, all cells from the connective tissue end up in a similar fibroblastoid cell type marked by loss of the specific expression pattern. It is still under discussion whether different de-differentiated mesenchymal cells have similar or identical differentiation capacities in vitro.\n\nMethodsChondrocytes isolated from patients with late-stage osteoarthritis were cultured for several passages until de-differentiation was completed. The mRNA level of cartilage markers was investigated, and the adipogenic, osteogenic and chondrogenic differentiation capacity was examined. By adding 5-aza-2-deoxycytidine (5-aza-dC) to the media, the influence of DNA methylation on the differentiation capacity was analyzed.\n\nResultsThe chondrocytes used in this work were not affected by the loss of specific gene expression upon cell culture. The mRNA levels of SOX5, SOX6, SOX9, aggrecan, and proteoglycan-4 remained unchanged. The underlying mechanisms of cartilage marker maintenance in osteoarthritic (OA) chondrocytes were investigated with a focus on the epigenetic modification by DNA methylation. The treatment of de-differentiated chondrocytes with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine (5-aza-dC) displayed no appreciable impact on the observed maintenance of marker gene expression, while the chondrogenic differentiation capacity was compromised. On the other hand, the pre-cultivation with 5-aza-dC improved the osteogenesis and adipogenesis of OA chondrocytes. Contradictory to these effects, the DNA methylation levels were not reduced after treatment with 1 M 5-aza-dC for four weeks.\n\nConclusionChondrocytes isolated from late-stage osteoarthritic patients represents a reliable cell source for in vitro studies as wells as disease models since the chondrogenic differentiation potential remains. 5-aza-2-deoxycytidine could not further improve their chondrogenic potential.

cell biology↗

bioRxiv2018

Isolation and characterization of extracellular vesicles from Caenorhabditis elegans for multi-omic analysis.

Cells from bacteria to human release vesicles into their extracellular environment. These extracellular vesicles (EVs) contain multiple classesof molecules, including nucleic acids, proteins, and lipids. The isolation and analysis of EV cargos from mammalian cell culture and liquid biopsysamples has become a powerful approach for uncovering the messages that are packaged into these organelles. However, this approach has not been tenable in invertebrate model systems due to lack of sufficient amounts of pure EVs. Here we report a robust and reproducible procedure to isolateEVs from Caenorhabditis elegans with yields similar to those obtained from human cell culture. Through nanoparticle tracking, transmission electron microscopy, flow cytometry, mass spectrometry, RNAseq, and immunoaffinity analysis we provide the first ever detailed characterization of C. elegans EV composition and demonstrate that C. elegans EVs share fundamentally similar properties with their mammalian counterparts. These include vesicle size, enrichment for lipid rafts, and similar types of RNA and protein cargos. This ability of isolate pure EVs on ascale amenable to multiple types of downstream analyses permits, multi-omics characterization of EV cargos in an invertebrate model system.

cell biology↗

bioRxiv2018

Realtime 2-5A kinetics suggests interferons β and {lambda} evade global arrest of translation by RNase L

Cells of all mammals recognize double-stranded RNA (dsRNA) as a foreign material. In response, they release interferons (IFNs) and activate a ubiquitously expressed pseudokinase/endoribonuclease RNase L. RNase L executes regulated RNA decay and halts global translation. Here we developed a biosensor for 2,5-oligoadenylate (2-5A), the natural activator of RNase L. We found that 2-5A was acutely synthesized by cells in response to dsRNA sensing, which immediately triggered cellular RNA cleavage by RNase L and arrested host protein synthesis. However, translation-arrested cells still transcribed IFN-stimulated genes (ISGs) and secreted IFNs of types I and III (IFN-{beta} and IFN-{lambda}). Our data suggests that IFNs escape from the action of RNase L on translation. We propose that 2-5A/RNase L pathway serves to rapidly and accurately suppress basal protein synthesis, preserving privileged production of defense proteins of the innate immune system.\n\nSignificanceRNase L is a mammalian enzyme that can stop global protein synthesis during interferon response. Cells must balance the need to make interferons (which are proteins) with the risk to lose cell-wide translation due to RNase L. This balance can most simply be achieved if RNase L was activated late in the interferon response. However, we show by engineering a biosensor for the RNase L pathway, that on the contrary, RNase L activation precedes interferon synthesis. Further, translation of interferons evades the action of RNase L. Our data suggest that RNase L facilitates a switch of protein synthesis from homeostasis to specific needs of innate immune signaling.

cell biology↗