Evaluating the Stability of mRNAs and Noncoding RNAs

Abstract

Changes in RNA stability have an important impact in the gene expression regulation. Different methods based on the transcription blockage with RNA polymerase inhibitors or metabolic labeling of newly synthesized RNAs have been developed to evaluate RNA decay rates in cultured cell. Combined with techniques to measure transcript abundance genome-wide, these methods have been used to reveal novel features of the eukaryotic transcriptome. The stability of protein-coding mRNAs is in general closely associated to the physiological function of their encoded proteins, with short-lived mRNAs being significantly enriched among regulatory genes whereas genes associated with housekeeping functions are predominantly stable. Likewise, the stability of noncoding RNAs (ncRNAs) seems to reflect their functional role in the cell. Thus, investigating RNA stability can provide insights regarding the function of yet uncharacterized regulatory ncRNAs. In this chapter, we discuss the methodologies currently used to estimate RNA decay and outline an experimental protocol for genome-wide estimation of RNA stability of protein-coding and lncRNAs. This protocol details the transcriptional blockage of cultured cells with actinomycin D, followed by RNA isolation at different time points, the determination of transcript abundance by qPCR/DNA oligoarray hybridization, and the calculation of individual transcript half-lives.

Identification of TMEM208 and PQLC2 as reference genes for normalizing mRNA expression in colorectal cancer treated with aspirin

“…Moreover, we detected that if aspirin treatment affected the mRNA decay of TMEM208 and PQLC2 in colon cancer cells. HCT116 cells were treated with and without 5 mM aspirin for 24 h, and then further treated with 5 μM Dactinomycin D, an inhibitor of RNA polymerase, for 0 h, 0.5 h, 1 h and 2 h respectively [29–31]. Finally, the relative ratio of TMEM208 to PQLC2 in cells with aspirin and without aspirin treatment at different time points after Dactinomycin D treatment was calculated respectively, and the result showed that the relative ratio of TMEM208 to PQLC2 was identical in HCT116 cells with and without aspirin treatment at each time point after Dactinomycin D treatment (Figure 6C), which suggests that aspirin treatment does not affect the mRNA decay of TMEM208 and PQLC2.…”

Peer Review #2 of "Circular RNAs as potential biomarkers and therapeutics for cardiovascular disease (v0.1)"

“…They are more abundant than expected and more stable than linear RNAs due to the covalently closed loop structures, which can resist RNA exonuclease and RNase R activity. In addition, the half-life of stable ncRNAs is about 48 hours, which is much more than that of mRNAs (10 hours) (Ayupe & Reis, 2017).…”

Regulatory Mechanism and Application of lncRNAs in Poultry

“…Natural antisense transcripts, Nats, also called natural antisense RNA, is a complementary transcription product of sense RNA produced in natural organisms, which is transcribed by antisense strand in DNA double strand and processed by RNA polymerase II (RNA polymerase II) [55][56][57][58][59]. NATs can regulate the expression of mRNA and protein at the transcriptional and post-transcriptional levels [60].…”