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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.

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.

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. SARS-CoV-2 in patient samples is detected in under an hour using a CRISPR-based lateral flow assay.

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.Structure maps of RNAs localized to chromatin, nucleoplasm and cytoplasm provide a rich resource with which to investigate the interplay of RNA structure, RNA–protein interactions and RNA modifications.

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.

The authors assess the role of N 6 -methyladenosine in T cell development and function, and show that RNA methylation controls T cell homeostasis by regulating IL-7-mediated STAT5 activation. A role for mRNA modification in T cell homeostasis N 3 -methyladenosine is a common modification of messenger RNA responsible for the regulation of mRNA metabolism such as turnover and stability. Here, Richard Flavell and colleagues assess its physiological role in T cell development and function, and show that RNA methylation controls T cell homeostasis by regulating IL-7-mediated STAT5 activation. N 6 -methyladenosine (m 6 A) is the most common and abundant messenger RNA modification, modulated by ‘writers’, ‘erasers’ and ‘readers’ of this mark 1 , 2 . In vitro data have shown that m 6 A influences all fundamental aspects of mRNA metabolism, mainly mRNA stability, to determine stem cell fates 3 , 4 . However, its in vivo physiological function in mammals and adult mammalian cells is still unknown. Here we show that the deletion of m 6 A ‘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 m 6 A, 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 m 6 A 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 m 6 A modification in T-cell-mediated pathogenesis and reveals a novel mechanism of T cell homeostasis and signal-dependent induction of mRNA degradation.

Dear Editor, N6-methyladenosine （m^6A） is the most abundant mRNA chemical modification, and is modulated by m^6A ＇writers＇, ＇erasers＇ and ＇readers＇ proteins . In vitro experiments suggest that m^6A regulates several aspects of RNA metabolism, including RNA decay, splicing and translation .

N6 -methyladenosine (m6A) is the most common and abundant messenger RNA modification, modulated by ‘writers’, ‘erasers’ and ‘readers’ of this mark 1,2. In vitro data have shown that m6A influences all fundamental aspects of mRNA metabolism, mainly mRNA stability, to determine stem cell fates 3,4. However, its in vivo physiological function in mammals and adult mammalian cells is still unknown. Here we show that deletion of m6A ‘writer’ protein METTL3 in mouse T cells disrupts T cell homeostasis and differentiation. In a lymphopenic mouse adoptive transfer model, naive Mettl3 deficient T cells failed to undergo homeostatic expansion and remarkably remained in the naïve state up through 12 weeks, thereby preventing colitis. Consistent with these observations, the mRNAs of SOCS family genes encoding STAT- signaling inhibitory proteins, Socs1, Socs3 and Cish, were marked by m6A, exhibited slower mRNA decay and increased mRNAs and protein expression levels in Mettl3 deficient naïve 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 plays important roles for inducible degradation of Socs mRNAs in response to IL-7 signaling in order to reprogram Naïve T cells for proliferation and differentiation. Our study elucidates for the first time 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.

MicroRNAs (miRNA) are critical regulators of development, cell differentiation, and the stress response. Mature miRNAs are small (~22 nucleotides) RNA molecules that post-transcriptionally regulate their targets by acting as guides for Argonaute (AGO) proteins. Despite the importance of these small RNAs in many pathways, the rules of miRNA target site recognition remain unclear. Currently, nucleotides 2-8 of the miRNA, termed the ‘seed’ sequence, are known to be critical for miRNA targeting. However, it is unclear if families of miRNAs, which share the same seed sequence, functionally target the same target sites. To improve our understanding of miRNA targeting, I generated a unique and reproducible dataset through the ligation of miRNAs to their target sites using individual nucleotide-resolution crosslinking and immunoprecipitation (iCLIP) in the nematode worm Caenorhabditis elegans, which is detailed in Chapter 2. As reviewed in Chapter 3, these ligated RNAs, called miRNA-target chimeras, provide biochemical evidence of which miRNAs are bound at a specific target site. Chapter 4 details my analysis of these chimeric data. Interestingly, the majority of miRNA target sites had the potential to support 3’ end interactions. In contrast to my prediction that these highly related miRNAs would bind the same sets of targets, family members from multiple miRNA families primarily targeted specific sets of transcripts. To confirm the importance of the 3’ end in vivo, I carried out in vivo rescue experiments that demonstrate that seed pairing is insufficient to mediate targeting in the absence of additional complementarity to nucleotides in the 3’ end of the miRNA. To confirm that 3’ end interactions direct binding specificity, I developed a novel method called ChimeraPCR (ChimP) that allowed for the detection of miRNA-target chimeras without the need for analyzing sequencing datasets. To understand the role of AGO proteins in regulating lifespan in C. elegans, I generated several genetic tools, which are described in Chapter 5. These tools facilitated studies of how the highly related AGO proteins, ALG-1 and ALG-2, have opposing roles in controlling lifespan.