The Architecture of SARS-CoV-2 Transcriptome.

SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic. Although the SARS-CoV-2 genome was reported recently, its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we 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 discontinuous transcription events. In addition to the canonical genomic and 9 subgenomic RNAs, SARS-CoV-2 produces 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, AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the modification and the 3' tail. Functional investigation of the unknown transcripts and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.

The Short- and Long-Range RNA-RNA Interactome of SARS-CoV-2.

The Coronaviridae is a family of positive-strand RNA viruses that includes SARS-CoV-2, the etiologic agent of the COVID-19 pandemic. Bearing the largest single-stranded RNA genomes in nature, coronaviruses are critically dependent on long-distance RNA-RNA interactions to regulate the viral transcription and replication pathways. Here we experimentally mapped the in vivo RNA-RNA interactome of the full-length SARS-CoV-2 genome and subgenomic mRNAs. We uncovered a network of RNA-RNA interactions spanning tens of thousands of nucleotides. These interactions reveal that the viral genome and subgenomes adopt alternative topologies inside cells and engage in different interactions with host RNAs. Notably, we discovered a long-range RNA-RNA interaction, the FSE-arch, that encircles the programmed ribosomal frameshifting element. The FSE-arch is conserved in the related MERS-CoV and is under purifying selection. Our findings illuminate RNA structure-based mechanisms governing replication, discontinuous transcription, and translation of coronaviruses and will aid future efforts to develop antiviral strategies.

Using All-Atom Potentials to Refine RNA Structure Predictions of SARS-CoV-2 Stem Loops.

A considerable amount of rapid-paced research is underway to combat the SARS-CoV-2 pandemic. In this work, we assess the 3D structure of the 5' untranslated region of its RNA, in the hopes that stable secondary structures can be targeted, interrupted, or otherwise measured. To this end, we have combined molecular dynamics simulations with previous Nuclear Magnetic Resonance measurements for stem loop 2 of SARS-CoV-1 to refine 3D structure predictions of that stem loop. We find that relatively short sampling times allow for loop rearrangement from predicted structures determined in absence of water or ions, to structures better aligned with experimental data. We then use molecular dynamics to predict the refined structure of the transcription regulatory leader sequence (TRS-L) region which includes stem loop 3, and show that arrangement of the loop around exchangeable monovalent potassium can interpret the conformational equilibrium determined by in-cell dimethyl sulfate (DMS) data.

Differences of time-dependent microRNA expressions in breast cancer cells.

MicroRNA (miRNA) expression is a dynamic process in the cell, and the proper time period for post-transcriptional regulation might be critical due to the gene-on/-off expression times of the cell. Here, we investigated the effect of different time-points on proliferation, invasion and miRNA expression profiles of human breast cancer cell lines MCF-7 (non-metastatic, epithelium-like breast cancer cell line with oestrogen receptor (ER) positive (+) and human breast cancer cell lines MDA-MB-435 (metastatic, invasive, ER negative (-). For this purpose, MCF-7 and MDA-MB-435 cells were seeded different number in E-plate 16 for proliferation experiment using an electrical impedance-based real-time cell analyzer system (RTCA) for 168 h. Similarly, invasion potential of MCF-7 and MDA-MB-435 were determined by RTCA for 90 h. Total RNAs including miRNAs were isolated at 2, 4, 6, 12, 24, 48 h from the MCF-7 and MDA-MB-435 cells. Afterward, the quantitative 84 miRNA expressions of MCF-7 and MDA-MB-435 were analyzed by Fluidigm Microfluidic 96.96 Dynamic Array. The results of these study demonstrated that both proliferation potential and invasion capacity of MDA-MB-435 is higher than MCF-7 as time-dependent manner. Furthermore, we detected that up/down expressions of 32 miRNAs at all time points in MDA-MB-435 compared to MCF-7 (at least ten-fold increased). Because of the high number of miRNAs, we more closely evaluated the expression of six of them (miR-100-5p, miR-29a-3p, miR-130a-3p, miR-10a-5p, miR-10b-5p, miR-203a), and determined that their levels were dramatically changed by at least 50-fold at different time points of the experiment (p < 0.01). The expression levels of five of these miRNAs (miR-100-5p, miR-10a-5p, miR-10b-5p, miR-130a-3p, and miR-29a-3p) started to increase from the fourth hour and continued to increase until the 48th hour in MDA-MB-435 cells compared to MCF-7 cells (p < 0.01). Simultaneously, the expression of one of these miRNAs (miR-203a) decreased from the sixth hour to the 48th hour in MDA-MB-435 as compared to MCF-7. We determined pathways associated with target genes using mirPath - DIANA TOOLS. Small RNAs including miRNA are essential regulatory molecules for gene expressions. In the literature, gene expressions have been published as burst and pulse in the form of discontinuous transcription. The data of the research suggested that time-dependent changes of miRNA expressions can be affected target gene transcriptional fluctuations in breast cancer cell and can be base for the further studies.

Transcriptional and epi-transcriptional dynamics of SARS-CoV-2 during cellular infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses subgenomic RNA (sgRNA) to produce viral proteins for replication and immune evasion. We apply long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA upregulates earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of open reading frame 1ab (ORF1ab) containing nsp1 joins to ORF10, and the 3' untranslated region (UTR) upregulates at 48 h post-infection in human cell lines. We identify double-junction sgRNA containing both TRS-dependent and -independent junctions. We find multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA and that sgRNA modifications are stable across transcript clusters, host cells, and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle.