The most widely used approach for defining gene function is to reduce or completely disrupt its normal expression. For over a decade, RNAi has ruled the lab, offering a magic bullet to disrupt gene ...expression in many organisms. However, new biotechnological tools—specifically CRISPR-based technologies—have become available and are squeezing out RNAi dominance in mammalian cell studies. These seemingly competing technologies leave research investigators with the question: “Which technology should I use in my experiment?” This review offers a practical resource to compare and contrast these technologies, guiding the investigator when and where to use this fantastic array of powerful tools.
Boettcher and McManus provide a practical guide to choosing the right tool for your gene silencing experiment—RNAi, TALEN, or CRISPR.
Known protein coding gene exons compose less than 3% of the human genome. The remaining 97% is largely uncharted territory, with only a small fraction characterized. The recent observation of ...transcription in this intergenic territory has stimulated debate about the extent of intergenic transcription and whether these intergenic RNAs are functional. Here we directly observed with a large set of RNA-seq data covering a wide array of human tissue types that the majority of the genome is indeed transcribed, corroborating recent observations by the ENCODE project. Furthermore, using de novo transcriptome assembly of this RNA-seq data, we found that intergenic regions encode far more long intergenic noncoding RNAs (lincRNAs) than previously described, helping to resolve the discrepancy between the vast amount of observed intergenic transcription and the limited number of previously known lincRNAs. In total, we identified tens of thousands of putative lincRNAs expressed at a minimum of one copy per cell, significantly expanding upon prior lincRNA annotation sets. These lincRNAs are specifically regulated and conserved rather than being the product of transcriptional noise. In addition, lincRNAs are strongly enriched for trait-associated SNPs suggesting a new mechanism by which intergenic trait-associated regions may function. These findings will enable the discovery and interrogation of novel intergenic functional elements.
Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in ...microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.
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► Ultracomplex shRNA library minimizes false positives/negatives in genome-wide screens ► Pooled double-shRNA strategy systematically maps genetic interactions between hits ► Application of two-step strategy identifies pathways controlling ricin susceptibility ► The resulting map uncovers functionally distinct mammalian TRAPP complexes
A high-throughput method that relies on the use of ultracomplex shRNA libraries makes it possible to create genetic interaction maps in mammalian cells. This approach will be applicable to many cellular processes and conditions, as illustrated by the discovery of distinct TRAPP complexes involved in endocytosis.
Acquired drug resistance prevents cancer therapies from achieving stable and complete responses. Emerging evidence implicates a key role for non-mutational drug resistance mechanisms underlying the ...survival of residual cancer 'persister' cells. The persister cell pool constitutes a reservoir from which drug-resistant tumours may emerge. Targeting persister cells therefore presents a therapeutic opportunity to impede tumour relapse. We previously found that cancer cells in a high mesenchymal therapy-resistant cell state are dependent on the lipid hydroperoxidase GPX4 for survival. Here we show that a similar therapy-resistant cell state underlies the behaviour of persister cells derived from a wide range of cancers and drug treatments. Consequently, we demonstrate that persister cells acquire a dependency on GPX4. Loss of GPX4 function results in selective persister cell ferroptotic death in vitro and prevents tumour relapse in mice. These findings suggest that targeting of GPX4 may represent a therapeutic strategy to prevent acquired drug resistance.
Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulating levels of BCAA are linked to obesity and ...diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.
Candidate enhancers can be identified on the basis of chromatin modifications, the binding of chromatin modifiers and transcription factors and cofactors, or chromatin accessibility. However, ...validating such candidates as bona fide enhancers requires functional characterization, typically achieved through reporter assays that test whether a sequence can increase expression of a transcriptional reporter via a minimal promoter. A longstanding concern is that reporter assays are mainly implemented on episomes, which are thought to lack physiological chromatin. However, the magnitude and determinants of differences in cis-regulation for regulatory sequences residing in episomes versus chromosomes remain almost completely unknown. To address this systematically, we developed and applied a novel lentivirus-based massively parallel reporter assay (lentiMPRA) to directly compare the functional activities of 2236 candidate liver enhancers in an episomal versus a chromosomally integrated context. We find that the activities of chromosomally integrated sequences are substantially different from the activities of the identical sequences assayed on episomes, and furthermore are correlated with different subsets of ENCODE annotations. The results of chromosomally based reporter assays are also more reproducible and more strongly predictable by both ENCODE annotations and sequence-based models. With a linear model that combines chromatin annotations and sequence information, we achieve a Pearson's R
of 0.362 for predicting the results of chromosomally integrated reporter assays. This level of prediction is better than with either chromatin annotations or sequence information alone and also outperforms predictive models of episomal assays. Our results have broad implications for how cis-regulatory elements are identified, prioritized and functionally validated.
Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived ...macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe−/− mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery.
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•M2 macrophage exosomes reprogram inflammation and energy metabolism in recipient cells•Specific miRNAs enriched in M2 macrophage exosomes reduce TNF-α and NF-κB signaling•M2 exosomes control atherosclerosis by reducing hematopoiesis and myeloid inflammation
Anti-inflammatory properties of M2 macrophages can be communicated as miRNA packaged into exosomes. Bouchareychas et al. show that when tested in the Apoe−/− mouse model of hyperlipidemia, M2 macrophage exosomes reduced hematopoiesis and the inflammatory state of circulating monocytes and macrophages in atherosclerotic plaques.
Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal ...directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair. Based on the results from over 100,000 perturbed gene pairs, we reconstruct a directional dependency network for human K562 leukemia cells and demonstrate how our approach allows the determination of directionality in activating genetic interactions. Our interaction network connects previously uncharacterized genes to well-studied pathways and identifies targets relevant for therapeutic intervention.
Assembly of microRNA ribonucleoproteins (miRNPs) or RNA-induced silencing complexes (RISCs) is essential for the function of miRNAs and initiates from processing of precursor miRNAs (pre-miRNAs) by ...Dicer or by Ago2. Here, we report an in vitro miRNP/RISC assembly assay programmed by pre-miRNAs from mammalian cell lysates. Combining in vivo studies in Dicer Knockout cells reconstituted with wild-type or catalytically inactive Dicer, we find that the miRNA loading complex (miRLC) is the primary machinery linking pre-miRNA processing to miRNA loading. We show that a miRNA precursor deposit complex (miPDC) plays a crucial role in Dicer-independent miRNA biogenesis and promotes miRNP assembly of certain Dicer-dependent miRNAs. Furthermore, we find that 5′-uridine, 3′-mid base pairing, and 5′-mid mismatches within pre-miRNAs promote their assembly into miPDC. Our studies provide a comprehensive view of miRNP/RISC assembly pathways in mammals, and our assay provides a versatile platform for further mechanistic dissection of such pathways in mammals.
► Development of in vitro miRNP/RISC assembly assay programmed by pre-miRNAs ► miRLC is the primary machinery that links pre-miRNA processing to miRNA loading ► Roles of miPDC in miRNA biogenesis ► 5′-U, 3′-mid base pairs, 5′-mid mismatches in pre-miRNAs promote miPDC assembly
Body temperature control is essential for survival. In mammals, thermoregulation is mediated by the preoptic area of anterior hypothalamus (POA), with ∼30% of its neurons sensitive to brain ...temperature change. It is still unknown whether and how these temperature-sensitive neurons are involved in thermoregulation, because for eight decades they have only been identified via electrophysiological recording. By combining single-cell RNA-seq with whole-cell patch-clamp recordings, we identified Ptgds as a genetic marker for temperature-sensitive POA neurons. Then, we demonstrated these neurons’ role in thermoregulation via chemogenetics. Given that Ptgds encodes the enzyme that synthesizes prostaglandin D2 (PGD2), we further explored its role in thermoregulation. Our study revealed that rising temperature of POA alters the activity of Ptgds-expressing neurons so as to increase PGD2 production. PGD2 activates its receptor DP1 and excites downstream neurons in the ventral medial preoptic area (vMPO) that mediates body temperature decrease, a negative feedback loop for thermoregulation.
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•PGD2 synthase is a marker for temperature-sensitive neurons in preoptic area (POA)•POA neurons that express PGD2 synthase are involved in thermoregulation•Rising POA temperature increases neuronal firing to promote PGD2 production•PGD2 activates the DP1 receptor in ventral medial POA neurons that mediate hypothermia
Single-cell RNA-seq combined with whole-cell patch-clamp recording identifies PGD2 synthase enriched in temperature-sensitive neurons in the medial preoptic area of mouse, which detects brain temperature increase and mediates body temperature decrease via PGD2 receptor DP1 expressed in ventral medial preoptic area.
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