The long noncoding RNA NEAT1 and nuclear paraspeckles are up-regulated by the transcription factor HSF1 in the heat shock response.

The long noncoding RNA (lncRNA) NEAT1 (nuclear enriched abundant transcript 1) is the architectural component of nuclear paraspeckles, and it has recently gained considerable attention as it is abnormally expressed in pathological conditions such as cancer and neurodegenerative diseases. NEAT1 and paraspeckle formation are increased in cells upon exposure to a variety of environmental stressors and believed to play an important role in cell survival. The present study was undertaken to further investigate the role of NEAT1 in cellular stress response pathways. We show that NEAT1 is a novel target gene of heat shock transcription factor 1 (HSF1) and is up-regulated when the heat shock response pathway is activated by sulforaphane (SFN) or elevated temperature. HSF1 binds specifically to a newly identified conserved heat shock element in the NEAT1 promoter. In line with this, SFN induced the formation of NEAT1-containing paraspeckles via an HSF1-dependent mechanism. HSF1 plays a key role in the cellular response to proteotoxic stress by promoting the expression of a series of genes, including those encoding molecular chaperones. We have found that the expression of HSP70, HSP90, and HSP27 is amplified and sustained during heat shock in NEAT1-depleted cells compared with control cells, indicating that NEAT1 feeds back via an unknown mechanism to regulate HSF1 activity. This interrelationship is potentially significant in human diseases such as cancer and neurodegenerative disorders.

CRISPR/Cas9 to Silence Long Non-Coding RNAs.

Knockout (KO) of long non-coding RNAs (lncRNAs) enables functional characterization of this still poorly described group of transcripts. One of the most efficient and simplest methods to achieve complete KO of a lncRNA is by employing CRISPR/Cas gene editing. As most lncRNAs are not well annotated, their individual functional regions are often not defined, and the majority of the transcripts are not affected by single nucleotide mutations. Therefore, CRISPR/Cas KO is more challenging for lncRNAs as compared to KO of protein coding genes. Strategies for lncRNAs KO include complete removal of the entire gene, removal of the promoter and transcriptional start site, abolishing exon-exon junctions, or removing the transcriptional termination site. Here, we describe the methodology to perform CRISPR/Cas9 KO of lncRNAs in vitro using electroporation as the method of transfection of presynthesized single guide RNAs (sgRNAs) and Cas9 enzyme.