Of mice and human-specific long noncoding RNAs

Abstract

The number of human LncRNAs has now exceeded all known protein-coding genes. Most studies of human LncRNAs have been conducted in cell culture systems where various mechanisms of action have been worked out. On the other hand, efforts to elucidate the function of human LncRNAs in an in vivo setting have been limited. In this brief review, we highlight some strengths and weaknesses of studying human LncRNAs in the mouse. Special consideration is given to bacterial artificial chromosome transgenesis and genome editing. The integration of these technical innovations offers an unprecedented opportunity to complement and extend the expansive literature of cell culture models for the study of human LncRNAs. Two different examples of how BAC transgenesis and genome editing can be leveraged to gain insight into human LncRNA regulation and function in mice are presented: the random integration of a vascular cell-enriched LncRNA and a targeted approach for a new LncRNA immediately upstream of the ACE2 gene, which encodes the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent underlying the coronavirus disease-19 (COVID-19) pandemic.

Fig. 1

Tissue RNA profile of SENCR and FLI1. Data obtained from the GTEx portal website (https://www.gtexportal.org/home/)

Fig. 2

ACE2 locus. Modified UCSC Genome Browser (http://genome.ucsc.edu/) screenshot showing the three isoforms of ACE2 and the upstream GS1-594A7.3 LncRNA. Vertebrate conservation (bottom green track) reveals conservation of ACE2 coding exons, but a notable lack of conservation in the two exons of GS1-594A7.3 (cream-colored rectangles overlapping exons)

Fig. 3

ACE2 protein expression and localization of GS1-594A7.3 LncRNA. A Western blotting shows ACE2 protein (molecular weight 120 kDa) in the indicated cell lines and human tissue types. B Cellular localization of GS1-594A7.3 LncRNA by two RNA-FISH methods, (i) ViewRNA which combines fluorescence in situ hybridization and sequential branched-DNA amplification in HEK-293 cells and (ii) biotin-labeled probes in Caco-2 cells pre-treated with a siRNA control (ii) or a siRNA targeting exon 2 of GS1-594A7.3 (iii). Scale bar is 10 µm. C Real-time quantitative PCR with standard curve to determine nuclear and cytoplasmic abundance of GS1-594A7.3 LncRNA with GAPDH as internal control

Fig. 4

Tissue RNA profile of ACE2 and GS1-594A7.3 LncRNA. Data obtained from the GTEx portal website (https://www.gtexportal.org/home/). Insets show heat maps of RNA expression for each transcript in human tissues

Fig. 5

Schematic of two methods of generating humanized BAC transgenic mice. Hypothetical LncRNA-mRNA gene pair within a human BAC (top). At least two methods exist for incorporating a BAC-containing LncRNA transgene into the mouse genome (arrows). One involves standard pronuclear injection with attending random integration, often as multiple copies (right arrow). An alternative method is recombinase-mediated genomic replacement (RMGR) (left arrow). Features of each method are indicated at bottom. See text for details