Cytoplasmic mRNA movements ultimately determine the spatial distribution of protein synthesis. Although some mRNAs are compartmentalized in cytoplasmic regions, most mRNAs, such as housekeeping mRNAs or the poly-adenylated mRNA population, are believed to be distributed throughout the cytoplasm. The general mechanism by which all mRNAs may move, and how this may be related to localization, is unknown. Here, we report a method to visualize single mRNA molecules in living mammalian cells, and we report that, regardless of any specific cytoplasmic distribution, individual mRNA molecules exhibit rapid and directional movements on microtubules. Importantly, the beta-actin mRNA zipcode increased both the frequency and length of these movements, providing a common mechanistic basis for both localized and nonlocalized mRNAs. Disruption of the cytoskeleton with drugs showed that microtubules and microfilaments are involved in the types of mRNA movements we have observed, which included complete immobility and corralled and nonrestricted diffusion. Individual mRNA molecules switched frequently among these movements, suggesting that mRNAs undergo continuous cycles of anchoring, diffusion, and active transport.

Understanding gene expression requires the ability to follow the fate of individual molecules. Here we use a cellular system for monitoring messenger RNA (mRNA)expression to characterize the movement in real time of single mRNA-protein complexes (mRNPs) in the nucleus of living mammalian cells. This mobility was not directed but was governed by simple diffusion. Some mRNPs were partially corralled throughout the nonhomogenous nuclear environment, but no accumulation at subnuclear domains was observed. Following energy deprivation, energyindependent motion of mRNPs was observed in a highly ATP-dependent nuclear environment; movements were constrained to chromatin-poor domains and excluded by newly formed chromatin barriers. This observation resolves a controversy, showing that the energetic requirements of nuclear mRNP trafficking are consistent with a diffusional model. Recent technological developments have facilitated imaging of single RNA molecules in the cytoplasm of living cells (1). We have developed a cellular system in which the expression of a transgene array can be followed sequentially in single living cells, and we have previously analyzed the particular chromatin-related modifications occurring at this specific locus from a silenced state throughout its transcriptional activation (2). Here we use this system to address the mechanism by which individual mRNA transcripts move within the nucleoplasm after release from the transcription site.In this system, a genetic locus, its transcribed mRNAs, and the translated protein were rendered visible in cells after electroporation with the cyan fluorescent protein (CFP) or the red fluorescent protein (RFP)-lac repressor protein (marks the genomic locus), yellow fluorescent protein (YFP)-MS2 (labels the mRNA), and pTet-On (for transcriptional induction) (3). Transcriptional activation by doxycycline induced the unfolding of the integrated locus (4) and the recruitment of the YFP-MS2 protein to the locus as a result of the specific labeling of the nascent transcripts bearing the MS2 stem loops. Minutes after induction (15 to 30 min), the MS2 signal began accumulating in the nucleoplasm in a particulate pattern suggestive of mRNA-protein complexes (mRNPs). At later times (1 to 2 hours after induction), mRNPs were detected in the cytoplasm in conjunction with the Correspondence to: Robert H. Singer, rhsinger@aecom.yu.edu. The presence of the nascent RNAs at the site of transcription was verified using fluorescent in situ hybridization (FISH) on fixed cells with two different probes either to the MS2 repeats (located in the middle of the transcript) or to the β globin exon (3′ end). Both probes hybridized at the active locus, indicating that the complete pre-mRNA transcripts were retained at the transcription site before release ( fig. S2A to D). Colocalization of the mRNA signal (FISH) with the YFP signal demonstrated that the particles visualized in living cells were mRNPs (3). RNA quantification with single-molecule sensitivity was performed on deconvol...

Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.

Several genome-wide association studies (GWAS) have identified a genetic polymorphism associated with the gene locus for interleukin 28B (IL28B), a type III interferon (IFN), as a major predictor of clinical outcome in hepatitis C. Antiviral effects of the type III IFN family have previously been shown against several viruses, including hepatitis C virus (HCV), and resemble the function of type I IFN including utilization of the intracellular JAK-STAT pathway. Effects unique to IL28B that would distinguish it from IFN-α are not well defined. By analyzing the transcriptomes of primary human hepatocytes (PHH) treated with IFN-α or IL28B, we sought to identify functional differences between IFN-α and IL28B to better understand the roles of these cytokines in the innate immune response. Although our data did not reveal distinct gene signatures, we detected striking kinetic differences between IFN-α and IL28B stimulation for interferon stimulated genes (ISGs). While gene induction was rapid and peaked at 8 h of stimulation with IFN-α in PHH, IL28B produced a slower, but more sustained increase in gene expression. We confirmed these findings in the human hepatoma cell line Huh7.5.1. Interestingly, in HCV infected cells, the rapid response after stimulation with IFN-α was blunted, and the induction pattern resembled that caused by IL28B. In conclusion, we describe the kinetics of gene induction as being fundamentally different for stimulations with either IFN-α or IL28B in hepatocytes suggesting distinct roles of these cytokines within the immune response. Furthermore, we demonstrate that the observed differences are substantially altered by infection with the hepatitis C virus.