Dynamic changes in LINC00458/HBL1 lncRNA expression during hiPSC differentiation to cardiomyocytes.

Long non-coding RNAs (lncRNAs) constitute the largest and most diverse class of non-coding RNAs. They localize to the nucleus, cytoplasm, or both compartments, and regulate gene expression through various mechanisms at multiple levels. LncRNAs tend to evolve faster and present higher tissue- and developmental stage-specific expression than protein-coding genes. Initially considered byproducts of erroneous transcription without biological function, lncRNAs are now recognized for their involvement in numerous biological processes, such as immune response, apoptosis, pluripotency, reprogramming, and differentiation. In this study, we focused on Heart Brake lncRNA 1 (HBL1), a lncRNA recently reported to modulate the process of pluripotent stem cell differentiation toward cardiomyocytes. We employed RT-qPCR and high-resolution RNA FISH to monitor the expression and localization of HBL1 during the differentiation progression. Our findings indicate a significant increase in HBL1 expression during mesodermal and cardiac mesodermal stages, preceding an anticipated decrease in differentiated cells. We detected the RNA in discrete foci in both the nucleus and in the cytoplasm. In the latter compartment, we observed colocalization of HBL1 with Y-box binding protein 1 (YB-1), which likely results from an interaction between the RNA and the protein, as the two were found to be coimmunoprecipitated in RNP-IP experiments. Finally, we provide evidence that HBL1, initially reported as an independent lncRNA gene, is part of the LINC00458 (also known as lncRNA-ES3 or ES3) gene, forming the last exon of some LINC00458 splice isoforms.

Replication of Severe Acute Respiratory Syndrome Coronavirus 2 in Human Respiratory Epithelium.

Currently, there are four seasonal coronaviruses associated with relatively mild respiratory tract disease in humans. However, there is also a plethora of animal coronaviruses which have the potential to cross the species border. This regularly results in the emergence of new viruses in humans. In 2002, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged and rapidly disappeared in May 2003. In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as a possible threat to humans, but its pandemic potential so far is minimal, as human-to-human transmission is ineffective. The end of 2019 brought us information about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emergence, and the virus rapidly spread in 2020, causing an unprecedented pandemic. At present, studies on the virus are carried out using a surrogate system based on the immortalized simian Vero E6 cell line. This model is convenient for diagnostics, but it has serious limitations and does not allow for understanding of the biology and evolution of the virus. Here, we show that fully differentiated human airway epithelium cultures constitute an excellent model to study infection with the novel human coronavirus SARS-CoV-2. We observed efficient replication of the virus in the tissue, with maximal replication at 2 days postinfection. The virus replicated in ciliated cells and was released apically.IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged by the end of 2019 and rapidly spread in 2020. At present, it is of utmost importance to understand the biology of the virus, rapidly assess the treatment potential of existing drugs, and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on SARS-CoV-2.

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.