Large numbers of long noncoding RNAs have been discovered in recent years, but only a few have been characterized. NEAT1 (nuclear paraspeckle assembly transcript 1) is a mammalian long noncoding RNA that is important for the reproductive physiology of mice, cancer development, and the formation of subnuclear bodies termed paraspeckles. The two major isoforms of NEAT1 (3.7 kb NEAT1_1 and 23 kb NEAT1_2 in human) are generated from a common promoter and are produced through the use of alternative transcription termination sites. This gene structure has made the functional relationship between the two isoforms difficult to dissect.
Researchers from the University of Western Australia used CRISPR-Cas9 genome editing to create several different cell lines: total NEAT1 knockout cells, cells that only express the short form NEAT1_1, and cells with twofold more NEAT1_2. Using these reagents, they obtained evidence that NEAT1_1 is not a major component of paraspeckles. In addition, their data suggest NEAT1_1 localizes in numerous nonparaspeckle foci they termed “microspeckles,” which may carry paraspeckle-independent functions. This study highlights the complexity of lncRNA and showcases how genome editing tools are useful in dissecting the structural and functional roles of overlapping transcripts.
Switching between the production of NEAT1 isoforms through genomic editing
(A) A schematic representation of the CRISPR editing strategy to switch the transcription of NEAT1 toward the production of NEAT1_2 isoform instead of NEAT1_1. All known signals [CFIm and canonical poly(A) signal] that could contribute to the polyadenylation of NEAT1_1 were identified and mutated using a double nickase Cas9 system (sgRNA_IS1 and IS2) alongside donor repair template in a YFP expression vector. Primers used for genotyping screening are indicated by arrows. Only two heterozygous and no homozygous clones were identified. The graphic representation is not scaled. (B–D) RT-qPCRs comparing two heterozygous NEAT1 isoform switched lines (NEAT1_IS lines) with the WT lines. (B) Total NEAT1 levels, (C) NEAT1_2 levels, and (D) proportion of NEAT1_2 in total NEAT1 levels. All RT-qPCR were normalized to housekeeping gene RPLP0. P-values are one-tailed Student t-test from four biological replicates. Error bars represent mean ± SD. (E) Number of paraspeckles per cells and (F) their areas were counted and analyzed using nonparametric one-way ANOVA with multiple testing, n > 100 cells in each cell type, paraspeckle count in each cell was plotted as scatter plots, and mean ± SD were plotted.