N6-Methyladenosine Modification Controls Circular RNA Immunity.

Circular RNAs (circRNAs) are prevalent in eukaryotic cells and viral genomes. Mammalian cells possess innate immunity to detect foreign circRNAs, but the molecular basis of self versus foreign identity in circRNA immunity is unknown. Here, we show that N6-methyladenosine (m6A) RNA modification on human circRNAs inhibits innate immunity. Foreign circRNAs are potent adjuvants to induce antigen-specific T cell activation, antibody production, and anti-tumor immunity in vivo, and m6A modification abrogates immune gene activation and adjuvant activity. m6A reader YTHDF2 sequesters m6A-circRNA and is essential for suppression of innate immunity. Unmodified circRNA, but not m6A-modified circRNA, directly activates RNA pattern recognition receptor RIG-I in the presence of lysine-63-linked polyubiquitin chain to cause filamentation of the adaptor protein MAVS and activation of the downstream transcription factor IRF3. CircRNA immunity has considerable parallel to prokaryotic DNA restriction modification system that transforms nucleic acid chemical modification into organismal innate immunity.

Pairing beyond the Seed Supports MicroRNA Targeting Specificity.

To identify endogenous miRNA-target sites, we isolated AGO-bound RNAs from Caenorhabditis elegans by individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP), which fortuitously also produced miRNA-target chimeric reads. Through the analysis of thousands of reproducible chimeras, pairing to the miRNA seed emerged as the predominant motif associated with functional interactions. Unexpectedly, we discovered that additional pairing to 3' sequences is prevalent in the majority of target sites and leads to specific targeting by members of miRNA families. By editing an endogenous target site, we demonstrate that 3' pairing determines targeting by specific miRNA family members and that seed pairing is not always sufficient for functional target interactions. Finally, we present a simplified method, chimera PCR (ChimP), for the detection of specific miRNA-target interactions. Overall, our analysis revealed that sequences in the 5' as well as the 3' regions of a miRNA provide the information necessary for stable and specific miRNA-target interactions in vivo.

Growth-dependent heterogeneity in the DNA damage response in Escherichia coli.

In natural environments, bacteria are frequently exposed to sub-lethal levels of DNA damage, which leads to the induction of a stress response (the SOS response in Escherichia coli). Natural environments also vary in nutrient availability, resulting in distinct physiological changes in bacteria, which may have direct implications on their capacity to repair their chromosomes. Here, we evaluated the impact of varying the nutrient availability on the expression of the SOS response induced by chronic sub-lethal DNA damage in E. coli. We found heterogeneous expression of the SOS regulon at the single-cell level in all growth conditions. Surprisingly, we observed a larger fraction of high SOS-induced cells in slow growth as compared with fast growth, despite a higher rate of SOS induction in fast growth. The result can be explained by the dynamic balance between the rate of SOS induction and the division rates of cells exposed to DNA damage. Taken together, our data illustrate how cell division and physiology come together to produce growth-dependent heterogeneity in the DNA damage response.

m6A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways.

N6-methyladenosine (m6A) is the most common and abundant messenger RNA modification, modulated by 'writers', 'erasers' and 'readers' of this mark. In vitro data have shown that m6A influences all fundamental aspects of mRNA metabolism, mainly mRNA stability, to determine stem cell fates. However, its in vivo physiological function in mammals and adult mammalian cells is still unknown. Here we show that the deletion of m6A 'writer' protein METTL3 in mouse T cells disrupts T cell homeostasis and differentiation. In a lymphopaenic mouse adoptive transfer model, naive Mettl3-deficient T cells failed to undergo homeostatic expansion and remained in the naive state for up to 12 weeks, thereby preventing colitis. Consistent with these observations, the mRNAs of SOCS family genes encoding the STAT signalling inhibitory proteins SOCS1, SOCS3 and CISH were marked by m6A, exhibited slower mRNA decay and showed increased mRNAs and levels of protein expression in Mettl3-deficient naive T cells. This increased SOCS family activity consequently inhibited IL-7-mediated STAT5 activation and T cell homeostatic proliferation and differentiation. We also found that m6A has important roles for inducible degradation of Socs mRNAs in response to IL-7 signalling in order to reprogram naive T cells for proliferation and differentiation. Our study elucidates for the first time, to our knowledge, the in vivo biological role of m6A modification in T-cell-mediated pathogenesis and reveals a novel mechanism of T cell homeostasis and signal-dependent induction of mRNA degradation.

The m6A RNA demethylase FTO is a HIF-independent synthetic lethal partner with the VHL tumor suppressor.

Loss of the von Hippel-Lindau (VHL) tumor suppressor is a hallmark feature of renal clear cell carcinoma. VHL inactivation results in the constitutive activation of the hypoxia-inducible factors (HIFs) HIF-1 and HIF-2 and their downstream targets, including the proangiogenic factors VEGF and PDGF. However, antiangiogenic agents and HIF-2 inhibitors have limited efficacy in cancer therapy due to the development of resistance. Here we employed an innovative computational platform, Mining of Synthetic Lethals (MiSL), to identify synthetic lethal interactions with the loss of VHL through analysis of primary tumor genomic and transcriptomic data. Using this approach, we identified a synthetic lethal interaction between VHL and the m6A RNA demethylase FTO in renal cell carcinoma. MiSL identified FTO as a synthetic lethal partner of VHL because deletions of FTO are mutually exclusive with VHL loss in pan cancer datasets. Moreover, FTO expression is increased in VHL-deficient ccRCC tumors compared to normal adjacent tissue. Genetic inactivation of FTO using multiple orthogonal approaches revealed that FTO inhibition selectively reduces the growth and survival of VHL-deficient cells in vitro and in vivo. Notably, FTO inhibition reduced the survival of both HIF wild type and HIF-deficient tumors, identifying FTO as an HIF-independent vulnerability of VHL-deficient cancers. Integrated analysis of transcriptome-wide m6A-seq and mRNA-seq analysis identified the glutamine transporter SLC1A5 as an FTO target that promotes metabolic reprogramming and survival of VHL-deficient ccRCC cells. These findings identify FTO as a potential HIF-independent therapeutic target for the treatment of VHL-deficient renal cell carcinoma.

COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme.

Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K/Q/A, and N501Y mutations, at least one of which is found in nearly all major variants. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all targeted SNP mutations. An analysis pipeline for CRISPR-based SNP identification from 261 clinical samples yielded a SNP concordance of 97.3% and agreement of 98.9% (258 of 261) for SARS-CoV-2 lineage classification, using SARS-CoV-2 whole-genome sequencing and/or real-time RT-PCR as test comparators. We also showed that detection of the single E484A mutation was necessary and sufficient to accurately identify Omicron from other major circulating variants in patient samples. These findings demonstrate the utility of CRISPR-based DETECTR as a faster and simpler diagnostic method compared with sequencing for SARS-CoV-2 variant identification in clinical and public health laboratories.

Opposing roles of microRNA Argonautes during Caenorhabditis elegans aging.

Argonaute (AGO) proteins partner with microRNAs (miRNAs) to target specific genes for post-transcriptional regulation. During larval development in Caenorhabditis elegans, Argonaute-Like Gene 1 (ALG-1) is the primary mediator of the miRNA pathway, while the related ALG-2 protein is largely dispensable. Here we show that in adult C. elegans these AGOs are differentially expressed and, surprisingly, work in opposition to each other; alg-1 promotes longevity, whereas alg-2 restricts lifespan. Transcriptional profiling of adult animals revealed that distinct miRNAs and largely non-overlapping sets of protein-coding genes are misregulated in alg-1 and alg-2 mutants. Interestingly, many of the differentially expressed genes are downstream targets of the Insulin/ IGF-1 Signaling (IIS) pathway, which controls lifespan by regulating the activity of the DAF-16/ FOXO transcription factor. Consistent with this observation, we show that daf-16 is required for the extended lifespan of alg-2 mutants. Furthermore, the long lifespan of daf-2 insulin receptor mutants, which depends on daf-16, is strongly reduced in animals lacking alg-1 activity. This work establishes an important role for AGO-mediated gene regulation in aging C. elegans and illustrates that the activity of homologous genes can switch from complementary to antagonistic, depending on the life stage.

Performance of Patient-Reported Outcomes Measurement Information System (PROMIS) scores compared with legacy metrics in evaluating outcomes after surgical treatment for osteochondritis dissecans of the humeral capitellum.

BACKGROUND: Patient-Reported Outcomes Measurement Information System (PROMIS) scores have not previously been used to measure long-term outcomes in operatively treated capitellar osteochondritis dissecans (OCD) patients. The aims of our study were to (1) assess patients' long-term outcomes using PROMIS scores, (2) compare the performance of PROMIS with other validated elbow legacy metrics, and (3) evaluate ceiling and floor effects in these outcome measures in patients undergoing surgical treatment for capitellar OCD. METHODS: We evaluated demographic characteristics, procedure details, preoperative PROMIS scores, and associated sports information in surgically treated pediatric capitellar OCD patients. An online survey was sent to the study participants, including the Kerlan-Jobe Orthopaedic Clinic (KJOC) shoulder and elbow score, the quick Disabilities of the Arm, Shoulder and Hand questionnaire, and the Liverpool Elbow Score patient-answered questionnaire, as well as the Mobility, Pain Interference, and Upper Extremity questionnaires from the PROMIS pediatrics bank. Correlations were evaluated between outcome measures. Ceiling and floor effects were evaluated for each outcome measure. RESULTS: Completed surveys were obtained for 57 patients (59 elbows). The mean patient age at surgery was 14 years (range, 10-18 years). The mean follow-up time was 6 years (standard deviation, 5 years; range, 1-18 years). The mean PROMIS Mobility score improved from 41.2 preoperatively to 55.2 postoperatively (P < .001). The mean Pain Interference score decreased from 46.9 preoperatively to 38 postoperatively (P < .001). The mean Upper Extremity score improved from 42.7 preoperatively to 53.2 postoperatively (P < .001). Significant correlations were observed between all legacy metrics and postoperative PROMIS scores (|r| > 0.54, P < .001). Ceiling or floor effects were seen in all legacy metrics and PROMIS scores. The KJOC score was least affected by ceiling or floor effects. CONCLUSION: There is a strong correlation between PROMIS scores and legacy measures evaluating outcomes after surgical management of capitellar OCD. However, large ceiling and floor effects were present in all measures, likely owing to the favorable clinical results. The KJOC score was limited the least by ceiling and floor effects.

A tale of two sequences: microRNA-target chimeric reads.

In animals, a functional interaction between a microRNA (miRNA) and its target RNA requires only partial base pairing. The limited number of base pair interactions required for miRNA targeting provides miRNAs with broad regulatory potential and also makes target prediction challenging. Computational approaches to target prediction have focused on identifying miRNA target sites based on known sequence features that are important for canonical targeting and may miss non-canonical targets. Current state-of-the-art experimental approaches, such as CLIP-seq (cross-linking immunoprecipitation with sequencing), PAR-CLIP (photoactivatable-ribonucleoside-enhanced CLIP), and iCLIP (individual-nucleotide resolution CLIP), require inference of which miRNA is bound at each site. Recently, the development of methods to ligate miRNAs to their target RNAs during the preparation of sequencing libraries has provided a new tool for the identification of miRNA target sites. The chimeric, or hybrid, miRNA-target reads that are produced by these methods unambiguously identify the miRNA bound at a specific target site. The information provided by these chimeric reads has revealed extensive non-canonical interactions between miRNAs and their target mRNAs, and identified many novel interactions between miRNAs and noncoding RNAs.

Identifying Argonaute binding sites in Caenorhabditis elegans using iCLIP.

The identification of endogenous targets remains an important challenge in understanding microRNA (miRNA) function. Past approaches using in silico methods and reporter constructs lack biological context that may enhance or inhibit target recognition. To address these limitations, several labs have utilized crosslinking and immunoprecipitation (CLIP) of Argonaute (Ago) proteins to identify miRNA targets. Recently, the Ule Lab introduced individual-nucleotide resolution CLIP (iCLIP) to increase the sensitivity of identifying protein-RNA interaction sites. Here we adapt the iCLIP protocol for use in Caenorhabditis elegans to identify endogenous sites targeted by the worm Argonaute (ALG-1) primarily responsible for miRNA function.

CRISPR-Cas enzymes: The toolkit revolutionizing diagnostics.

The programmable nature of sequence-specific targeting by CRISPR-Cas nucleases has revolutionized a wide range of genomic applications and is now emerging as a method for nucleic acid detection. We explore how the diversity of CRISPR systems and their fundamental mechanisms have given rise to a wave of new methods for target recognition and readout. These cross-disciplinary advances found at the intersection of CRISPR biology and engineering have led to the ability to rapidly generate solutions for emerging global challenges like the COVID-19 pandemic. We further discuss the advances and potential for CRISPR-based detection to have an impact across a continuum of diagnostic applications.

Rapid Detection of 2019 Novel Coronavirus SARS-CoV-2 Using a CRISPR-based DETECTR Lateral Flow Assay.

An outbreak of novel betacoronavirus, SARS-CoV-2 (formerly named 2019-nCoV), began in Wuhan, China in December 2019 and the COVID-19 disease associated with infection has since spread rapidly to multiple countries. Here we report the development of SARS-CoV-2 DETECTR, a rapid (~30 min), low-cost, and accurate CRISPR-Cas12 based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated this method using contrived reference samples and clinical samples from infected US patients and demonstrated comparable performance to the US CDC SARS-CoV-2 real-time RT-PCR assay.

CRISPR-Cas12-based detection of SARS-CoV-2.

An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation.

The Xist lncRNA mediates X chromosome inactivation (XCI). Here we show that Spen, an Xist-binding repressor protein essential for XCI , binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen loss activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing. ERV RNA and Xist A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in strictly local gene silencing in cis. These results suggest that Xist may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.

The genetic material inside cells is often packaged into thread-like structures called chromosomes. In humans, mice and other mammals, a pair of sex chromosomes determines the genetic or chromosomal sex of each individual. Those who inherit two "X" chromosomes are said to be chromosomally female, while chromosomal males have one "X" and one "Y" chromosome. This means females have twice as many copies of genes on the X chromosome as a male does, which turns out to be double the number that the body needs. To solve this problem, mammals have developed a strategy known as dosage compensation. The second X chromosome in females becomes "silent": its DNA remains unchanged, but none of the genes are active. A long noncoding RNA molecule called Xist is responsible for switching off the extra X genes in female cells. It does this by coating the entirety of the second X chromosome. Normally, RNA molecules transmit the coded instructions in genes to the cellular machinery that manufactures proteins. "Noncoding" RNAs like Xist, however, are RNAs that have taken on different jobs inside the cell. Researchers believe that the ancestral Xist gene may have once encoded a protein but changed over time to produce only a noncoding RNA. Carter, Xu et al. therefore set out to find out how exactly this might have happened, and also how Xist might have acquired its ability to switch genes off. Initial experiments used mouse cells grown in the laboratory, in which a protein called Spen was deleted. Spen is known to help Xist silence the X chromosome. In female cells lacking Spen, the second X chromosome remained active. Other chromosomes in male and female cells also had stretches of DNA that became active upon Spen's removal. These DNA sequences, termed endogenous retroviruses, were remnants of ancestral viral infections. In other words, Spen normally acted as an antiviral defense. Analysis of genetic sequences showed that Spen recognized endogenous retrovirus sequences resembling a key region in Xist, a region which was needed for Xist to work properly. Inserting fragments of endogenous retroviruses into a defective version of Xist lacking this region also partially restored its ability to inactivate genes, suggesting that X chromosome silencing might work by hijacking cellular defenses against viruses. That is, female cells essentially 'pretend' there is a viral infection on the second X chromosome by coating it with Xist (which mimics endogenous retroviruses), thus directing Spen to shut it down. This research is an important step towards understanding how female cells carry out dosage compensation in mammals. More broadly, it sheds new light on how ancient viruses may have shaped the evolution of noncoding RNAs in the human genome.

RNA structure maps across mammalian cellular compartments.

RNA structure is intimately connected to each step of gene expression. Recent advances have enabled transcriptome-wide maps of RNA secondary structure, called 'RNA structuromes'. However, previous whole-cell analyses lacked the resolution to unravel the landscape and also the regulatory mechanisms of RNA structural changes across subcellular compartments. Here we reveal the RNA structuromes in three compartments, chromatin, nucleoplasm and cytoplasm, in human and mouse cells. The cytotopic structuromes substantially expand RNA structural information and enable detailed investigation of the central role of RNA structure in linking transcription, translation and RNA decay. We develop a resource with which to visualize the interplay of RNA-protein interactions, RNA modifications and RNA structure and predict both direct and indirect reader proteins of RNA modifications. We also validate a novel role for the RNA-binding protein LIN28A as an N6-methyladenosine modification 'anti-reader'. Our results highlight the dynamic nature of RNA structures and its functional importance in gene regulation.

CELA2A mutations predispose to early-onset atherosclerosis and metabolic syndrome and affect plasma insulin and platelet activation.

Factors that underlie the clustering of metabolic syndrome traits are not fully known. We performed whole-exome sequence analysis in kindreds with extreme phenotypes of early-onset atherosclerosis and metabolic syndrome, and identified novel loss-of-function mutations in the gene encoding the pancreatic elastase chymotrypsin-like elastase family member 2A (CELA2A). We further show that CELA2A is a circulating enzyme that reduces platelet hyperactivation, triggers both insulin secretion and degradation, and increases insulin sensitivity. CELA2A plasma levels rise postprandially and parallel insulin levels in humans. Loss of these functions by the mutant proteins provides insight into disease mechanisms and suggests that CELA2A could be an attractive therapeutic target.

Detection of microRNA-Target Interactions by Chimera PCR (ChimP).

MicroRNAs (miRNAs) regulate gene expression by directing Argonaute proteins to target RNAs, which usually results in destabilization and translational inhibition of the target RNA. The prediction of animal miRNA target sites has remained a challenge due to the ability of miRNAs to bind target RNAs through imperfect base pairing. Recently, several labs have established methods to produce biochemical evidence of miRNA-target interactions by generating chimeric reads where the miRNA is ligated to its target RNA. Despite the insights that can be gained from chimera producing methods, the current approaches are inefficient, labor intensive and require computational expertise. Here we describe a method, called Chimera PCR (ChimP), for the validation or testing of specific miRNA-target interactions. This method allows for focused experiments to analyze miRNA targeting in a variety of conditions.

TRIM79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral RNA polymerase.

In response to virus infection, type I interferons (IFNs) induce several genes, most of whose functions are largely unknown. Here, we show that the tripartite motif (TRIM) protein, TRIM79α, is an IFN-stimulated gene (ISG) product that specifically targets tick-borne encephalitis virus (TBEV), a Flavivirus that causes encephalitides in humans. TRIM79α restricts TBEV replication by mediating lysosome-dependent degradation of the flavivirus NS5 protein, an RNA-dependent RNA polymerase essential for virus replication. NS5 degradation was specific to tick-borne flaviviruses, as TRIM79α did not recognize NS5 from West Nile virus (WNV) or inhibit WNV replication. In the absence of TRIM79α, IFN-ß was less effective in inhibiting tick-borne flavivirus infection of mouse macrophages, highlighting the importance of a single virus-specific ISG in establishing an antiviral state. The specificity of TRIM79α for TBEV reveals a remarkable ability of the innate IFN response to discriminate between closely related flaviviruses.