Despite the discovery of animal coronaviruses related to SARS-CoV-2, the evolutionary origins of this virus are elusive. We describe a meta-transcriptomic study of 411 bat samples collected from a small geographical region in Yunnan province, China, between May 2019 and November 2020. We identified 24 full-length coronavirus genomes, including four novel SARS-CoV-2 related and three SARS-CoV related viruses. Rhinolophus pusillus virus RpYN06 was the closest relative of SARS-CoV-2 in most of the genome, although it possessed a more divergent spike gene. The other three SARS-CoV-2 related coronaviruses carried a genetically distinct spike gene that could weakly bind to the hACE2 receptor in vitro . Ecological modeling predicted the co-existence of up to 23 Rhinolophus bat species, with the largest contiguous hotspots extending from South Laos and Vietnam to southern China. Our study highlights the remarkable diversity of bat coronaviruses at the local scale, including close relatives of both SARS-CoV-2 and SARS-CoV.

Activation of EGFR-Ras-MAPK signaling in vulval precursor cells (VPCs) by LIN-3/EGF from the gonad induces vulval development in C. elegans. The prevailing view is that LIN-3 overcomes an "inhibitory signal" from the adjacent hyp7 hypodermal syncytium. This view originated from observations indicating that inactivation of functionally redundant Synthetic Multivulva (SynMuv) genes in hyp7 can activate EGFR-Ras-MAPK signaling in the VPCs. Many SynMuv genes encode transcription and chromatin-associated factors, including the Rb ortholog. Here, we show that the SynMuv A and SynMuv B gene classes are functionally redundant for transcriptional repression of the key target gene, lin-3/EGF, in the hypodermis. These observations necessitate a revision of the concept of "inhibitory signaling." They also underscore the importance of preventing inappropriate cell signaling during development and suggest that derepression of growth factors may be the mechanism by which tumor suppressor genes such as Rb can have cell nonautonomous effects.

In Caenorhabditis elegans, vulval cell-fate specification involves the activities of multiple signal transduction and regulatory pathways that include a receptor tyrosine kinase/Ras/mitogen-activated protein kinase pathway and synthetic multivulva (SynMuv) pathways. Many genes in the SynMuv pathways encode transcription factors including the homologs of mammalian Rb, E2F, and components of the nucleosome-remodeling deacetylase complex. To further elucidate the functions of the SynMuv genes, we performed a genome-wide RNA interference (RNAi) screen to search for genes that antagonize the SynMuv gene activities. Among those that displayed a varying degree of suppression of the SynMuv phenotype, 32 genes are potentially involved in chromatin remodeling (called SynMuv suppressor genes herein). Genetic mutations of two representative genes (zfp-1 and mes-4) were used to further characterize their positive roles in vulval induction and relationships with Ras function. Our analysis revealed antagonistic roles of the SynMuv suppressor genes and the SynMuv B genes in germline-soma distinction, RNAi, somatic transgene silencing, and tissue specific expression of pgl-1 and the lag-2/Delta genes. The opposite roles of these SynMuv B and SynMuv suppressor genes on transcriptional regulation were confirmed in somatic transgene silencing. We also report the identifications of ten new genes in the RNAi pathway and six new genes in germline silencing. Among the ten new RNAi genes, three encode homologs of proteins involved in both protein degradation and chromatin remodeling. Our findings suggest that multiple chromatin remodeling complexes are involved in regulating the expression of specific genes that play critical roles in developmental decisions.

The retinoblastoma (Rb) tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.

Environmental stresses and nutrition availability critically affect animal development. Numerous animal species across multiple phyla enter developmental arrest for long-term survival in unfavorable environments and resume development upon stress removal. Here we show that compromising overall microRNA (miRNA) functions or mutating certain individual miRNAs impairs the longterm survival of nematodes during starvation-induced L1 diapause. We provide evidence that miRNA miR-71 is not required for the animals' entry into L1 diapause, but plays a critical role in long-term survival by repressing the expression of insulin receptor/PI3K pathway genes and genes acting downstream or in parallel to the pathway. Furthermore, miR-71 plays a prominent role in developmental recovery from L1 diapause partly through repressing the expression of certain heterochronic genes. The presented results indicate that interactions between multiple miRNAs and likely a large number of their mRNA targets in multiple pathways regulate the response to starvation-induced L1 diapause.F ood deprivation is a life-threatening challenge that animals frequently face as individuals and as species. Different organisms have developed versatile growth arrest strategies to overcome starvation-induced metabolic and developmental problems. The coordinated entrance into developmental arrest, long-term survival, and the reinitiation of development upon food availability are important biological processes to investigate.The nematode Caenorhabditis elegans responds to starvation by entering developmental arrest at multiple stages of its life cycle (1). When late, first larval stage (L1) worms sense unfavorable conditions, they enter an alternative and long-lived larval stage called dauer larvae (or dauer diapause). However, when newly hatched L1 worms encounter an environment with no food, developmental programs arrest and the worm enters L1 diapause. Unlike dauer diapause, L1 diapause is not accompanied by life cycle changes and has not been shown to require certain signaling pathways that control the formation of dauer diapause [such as TGF-β signaling (daf-1, daf-7) and nuclear hormone receptor (daf-12)] (2, 3). Furthermore, worms that are long-lived due to dietary restriction or decreased mitochondrial respiratory rates are short-lived during L1 diapause, suggesting that the mechanisms controlling L1 starvation survival are different at least in some aspects from those controlling aging (3).Previous studies showed that the release of postdocking calciumregulated dense-core vesicles, the insulin receptor (InsR) pathway, the AMPK pathway, and protein chaperones are required for the long-term survival of starved L1 worms (2-4). The roles of InsRs have also been implicated in arresting the cell cycle in germ cells and a portion of somatic cells during L1 diapause (2, 4). Upon entering L1 diapause, RNA polymerase II quickly accumulates and pauses at promoter regions, and this accumulation was speculated to stop transcription and facilitate the immediate reinit...

Null mutations in lin-35, the Caenorhabditis elegans ortholog of the mammalian Rb protein, cause no obvious morphological defects. Using a genetic approach to identify genes that may function redundantly with lin-35, we have isolated a mutation in the C. elegans psa-1 gene. lin-35; psa-1 double mutants display severe developmental defects leading to early larval arrest and adult sterility. The psa-1 gene has previously been shown to encode a C. elegans homolog of yeast SWI3, a critical component of the SWI/SNF complex, and has been shown to regulate asymmetric cell divisions during C. elegans development. We observed strong genetic interactions between psa-1 and lin-35 as well as a subset of the class B synMuv genes that include lin-37 and lin-9. Loss-of-function mutations in lin-35, lin-37, and lin-9 strongly enhanced the defects of asymmetric T cell division associated with a psa-1 mutation. Our results suggest that LIN-35/Rb and a certain class B synMuv proteins collaborate with the SWI/SNF protein complex to regulate the T cell division as well as other events essential for larval growth.

Although a variety of SARS-CoV-2 related coronaviruses have been identified, the evolutionary origins of this virus remain elusive. We describe a meta-transcriptomic study of 411 samples collected from 23 bat species in a small (~1100 hectare) region in Yunnan province, China, from May 2019 to November 2020. We identified coronavirus contigs in 40 of 100 sequencing libraries, including seven representing SARS-CoV-2-like contigs. From these data we obtained 24 full-length coronavirus genomes, including four novel SARS-CoV-2 related and three SARS-CoV related genomes. Of these viruses, RpYN06 exhibited 94.5% sequence identity to SARS-CoV-2 across the whole genome and was the closest relative of SARS-CoV-2 in the ORF1ab, ORF7a, ORF8, N, and ORF10 genes. The other three SARS-CoV-2 related coronaviruses were nearly identical in sequence and clustered closely with a virus previously identified in pangolins from Guangxi, China, although with a genetically distinct spike gene sequence. We also identified 17 alphacoronavirus genomes, including those closely related to swine acute diarrhea syndrome virus and porcine epidemic diarrhea virus. Ecological modeling predicted the co-existence of up to 23 Rhinolophus bat species in Southeast Asia and southern China, with the largest contiguous hotspots extending from South Lao and Vietnam to southern China. Our study highlights both the remarkable diversity of bat viruses at the local scale and that relatives of SARS-CoV-2 and SARS-CoV circulate in wildlife species in a broad geographic region of Southeast Asia and southern China. These data will help guide surveillance efforts to determine the origins of SARS-CoV-2 and other pathogenic coronaviruses.

RNA polymerase II (Pol II) transcription termination involves two linked processes: mRNA 3-end formation and release of Pol II from DNA. Signals for 3 processing are recognized by a protein complex that includes cleavage polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF). Here we identify suppressors encoding proteins that play roles in processes at the 3 ends of genes by exploiting a mutation in which the 3 end of another gene is transposed into the first gene of the Caenorhabditis elegans lin-15 operon. As expected, genes encoding CPSF and CstF were identified in the screen. We also report three suppressors encoding proteins containing a domain that interacts with the C-terminal domain of Pol II (CID). We show that two of the CID proteins are needed for efficient 3 cleavage and thus may connect transcription termination with RNA cleavage. Furthermore, our results implicate a serine/arginine-rich (SR) protein, SRp20, in events following 3-end cleavage, leading to termination of transcription.Caenorhabditis elegans operon ͉ RNA polymerase II C-terminal domain ͉ SRp20 ͉ CTD T ermination of RNA polymerase II (Pol II)-mediated transcription plays an important role in gene regulation and involves two linked processes: 3Ј-end formation and release of the Pol II from the DNA (1). Accordingly, termination of Pol II requires both the presence of an intact 3Ј-processing signal and several 3Ј-end processing factors including the cleavage and polyadenylation specificity factor (CPSF) and the cleavage stimulation factor (CstF) (2-6). Several of the polyadenylation factors are associated with the C-terminal domain (CTD) of the largest subunit of Pol II, which is known to be required for efficient 3Ј processing (7-10). Much is known about what is required for 3Ј-end processing, but which factors specifically act in transcription termination and how these factors cause the Pol II complex to terminate are not entirely clear. The known link between 3Ј-end formation and transcription termination has led to multiple models to explain transcription termination.Two such models have been proposed to explain the connection between 3Ј-end processing and transcription termination. One model, known as the ''allosteric'' model, proposes that termination is triggered by a conformational change of the Pol II complex that occurs on the emergence of the polyadenylation sequences (3). Another model, known as the ''torpedo'' model, proposes that termination is triggered subsequent to the cleavage event by the exonuclease XRN-2. When cleavage occurs at the poly(A) polymerase site, Pol II continues to transcribe a now-uncapped downstream RNA. This RNA is subject to degradation by the 5Ј-3Ј exonuclease XRN-2; according to the torpedo model, termination occurs when the exonuclease collides with Pol II (11-13). Recently, a unified allosteric-torpedo model has been proposed to explain new experimental evidence in support of both the earlier models (14,15).One commonality between the three models of termination is the obl...