The SARS-CoV-2 RNA interactome

SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its ability to repurpose host RNA-binding proteins (RBPs) to form its own RNA interactome. Here, we developed and applied a robust ribonucleoprotein capture protocol to uncover the SARS-CoV-2 RNA interactome. We report 109 host factors that directly bind to SARS-CoV-2 RNAs including general antiviral factors such as ZC3HAV1, TRIM25, and PARP12. Applying RNP capture on another coronavirus HCoV-OC43 revealed evolutionarily conserved interactions between viral RNAs and host proteins. Network and transcriptome analyses delineated antiviral RBPs stimulated by JAK-STAT signaling and proviral RBPs responsible for hijacking multiple steps of the mRNA life cycle. By knockdown experiments, we further found that these viral-RNA-interacting RBPs act against or in favor of SARS-CoV-2. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.

The architecture of SARS-CoV-2 transcriptome

SARS-CoV-2 is a betacoronavirus that is responsible for the COVID-19 pandemic. The genome of SARS-CoV-2 was reported recently, but its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we here present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical. In addition to the genomic RNA and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces a large number of transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif being AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the internal modification and the 3' tail. Functional investigation of the unknown ORFs and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2. HighlightsO_LIWe provide a high-resolution map of SARS-CoV-2 transcriptome and epitranscriptome using nanopore direct RNA sequencing and DNA nanoball sequencing. C_LIO_LIThe transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical. C_LIO_LIIn addition to the genomic and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces transcripts encoding unknown ORFs. C_LIO_LIWe discover at least 41 potential RNA modification sites with an AAGAA motif. C_LI

Development of a Laboratory-safe and Low-cost Detection Protocol for SARS-CoV-2 of the Coronavirus Disease 2019 (COVID-19)

The severe acute respiratory coronavirus 2 (SARS-CoV-2), which emerged in December 2019 in Wuhan, China, has spread rapidly to over a dozen countries. Especially, the spike of case numbers in South Korea sparks pandemic worries. This virus is reported to spread mainly through personto- person contact via respiratory droplets generated by coughing and sneezing, or possibly through surface contaminated by people coughing or sneezing on them. More critically, there have been reports about the possibility of this virus to transmit even before a virus-carrying person to show symptoms. Therefore, a low-cost, easy-access protocol for early detection of this virus is desperately needed. Here, we have established a real-time reverse-transcription PCR (rtPCR)-based assay protocol composed of easy specimen self-collection from a subject via pharyngeal swab, Trizolbased RNA purification, and SYBR Green-based rtPCR. This protocol shows an accuracy and sensitivity limit of 1-10 virus particles as we tested with a known lentivirus. The cost for each sample is estimated to be less than 15 US dollars. Overall time it takes for an entire protocol is estimated to be less than 4 hours. We propose a cost-effective, quick-and-easy method for early detection of SARS-CoV-2 at any conventional Biosafety Level II laboratories that are equipped with a rtPCR machine. Our newly developed protocol should be helpful for a first-hand screening of the asymptomatic virus-carriers for further prevention of transmission and early intervention and treatment for the rapidly propagating virus.

Gastric Carcinogenesis in the miR-222/221 Transgenic Mouse Model / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: MicroRNAs (miRNAs) regulate various cellular functions, including development, cell proliferation, apoptosis, and tumorigenesis. Different signatures associated with various tissue types, diagnosis, progression, prognosis, staging, and treatment response have been identified by miRNA expression profiling of human tumors. miRNAs function as oncogenes or as tumor suppressors. The relationship between gastric cancer and miRNA garnered attention due to the high incidence of gastric cancer in Asian countries. miR-222/221 expression increases in gastric tumor tissues. The oncogenic effect of miR-222/221 was previously determined in functional studies and xenograft models. In this study, transgenic mice over-expressing miR-222/221 were generated to confirm the effect of miR-222/221 on gastric carcinogenesis. MATERIALS AND METHODS: At 6 weeks of age, 65 transgenic mice and 53 wild-type mice were given drinking water containing N-nitroso-N-methylurea (MNU) for 5 alternating weeks to induce gastric cancer. The mice were euthanized at 36 weeks of age and histologic analysis was performed. RESULTS: Hyperplasia was observed in 3.77% of the wild-type mice and in 18.46% of the transgenic mice (p=0.020). Adenoma was observed in 20.75% of the wild-type mice and 26.15% of the transgenic mice (p=0.522). Carcinoma was observed in 32.08% of the wild-type mice and 41.54% of the transgenic mice (p=0.341). The frequency of hyperplasia, adenoma, and carcinoma was higher in transgenic mice, but the difference was statistically significant only in hyperplasia. CONCLUSION: These results suggest that hyperplasia, a gastric pre-cancerous lesion, is associated with miR-222/221 expression but miR-222/221 expression does not affect tumorigenesis itself.

Prediction of Mammalian MicroRNA Targets : Comparative Genomics Approach with Longer 3' UTR Databases

MicroRNAs play an important role in regulating gene expression, but their target identification is a difficult task due to their short length and imperfect complementarity. Burge and coworkers developed a program called TargetScan that allowed imperfect complementarity and established a procedure favoring targets with multiple binding sites conserved in multiple organisms. We improved their algorithm in two major aspects - (i) using well-defined UTR (untranslated region) database, (ii) examining the extent of conservation inside the 3' UTR specifically. Average length in our UTR database, based on the ECgene annotation, is more than twice longer than the Ensembl. Then, TargetScan was used to identify putative binding sites. The extent of conservation varies significantly inside the 3' UTR. We used the "tight" tracks in the UCSC genome browser to select the conserved binding sites in multiple species. By combining the longer 3' UTR data, TargetScan, and tightly conserved blocks of genomic DNA, we identified 107 putative target genes with multiple binding sites conserved in multiple species, of which 85 putative targets are novel.

RNA Interference in Functional Genomics and Medicine

RNA interference (RNAi) is the sequence-specific gene silencing induced by double-stranded RNA (dsRNA). Being a highly specific and efficient knockdown technique, RNAi not only provides a powerful tool for functional genomics but also holds a promise for gene therapy. The key player in RNAi is small RNA (~22-nt) termed siRNA. Small RNAs are involved not only in RNAi but also in basic cellular processes, such as developmental control and heterochromatin formation. The interesting biology as well as the remarkable technical value has been drawing widespread attention to this exciting new field.