Abstract
Regulation of gene expression by premature termination of transcription has been well described in all domains of life, including metazoans, yeast, plants, and bacteria. Although methods for identification of such regulatory events by sequencing are available, the focused biochemical studies of the mechanism are hampered by lack of highly sensitive and accurate experimental methods. Here, we propose a new method for absolute quantification of premature transcription termination events, PTT-quant. It is based on highly sensitive two-step digital droplet PCR protocol, coupled with normalized cDNA synthesis attained by site-specific pre-cleavage of investigated transcripts with RNase H. As a consequence, our method enables the reliable and sensitive quantification of both, prematurely terminated and full-length transcripts. By application of our method to investigation of transcriptional riboswitches in Bacillus subtilis, we were able to precisely measure the dynamics of S-adenosylmethionine (SAM) riboswitch induction, which turned to be ~ 23% higher in comparison the results obtained without cDNA synthesis normalization.Key points• A novel method for quantification of premature transcription termination events was established.• PTT-quant measures absolute concentration of full-length and terminated transcripts.• RNase H and the digital droplet PCR technique is used in PTT-quant.
Highlights
The premature transcription termination (PTT) has been discovered decades ago, but only recently, it has been recognized as one of the major regulatory mechanism, leading to the diversification of the gene expression
Among high sensitivity amplification-based techniques, the reverse-transcriptase digital droplet PCR (RT-Droplet digital PCR (ddPCR)) seems to be the best suited for the investigation of the PTT activity, due to the superior accuracy, without the need for the standard curve estimation, obtained by an absolute quantification of the nucleic acids. It enables the employment of the two-step protocol, consisting of an initial reverse-transcription of the total RNA with the employment of random hexamers, followed by the multiple target-specific ddPCR-based cDNA quantifications from a single cDNA sample
During the optimization of the RT-ddPCR workflow for the detection of dengue virus, they investigated the efficiency of the RT-ddPCR with the employment of the probes designed toward different regions of the viral RNA genome
Summary
The premature transcription termination (PTT) has been discovered decades ago, but only recently, it has been recognized as one of the major regulatory mechanism, leading to the diversification of the gene expression. The PTT has been reported in all kingdoms of life. Depending on the gene region where the termination occurs, two different types of the PTT can be distinguished. The transcription start site (TSS)-linked PTT is most likely related to the low efficiency of the full-length gene transcription by. (Singh et al 2018). Most of such transcripts are capped and polyadenylated, playing important roles in cell metabolism. The PTT has been shown to form mRNA variants encoding soluble isoforms of the transmembrane T-cell co-stimulator CD46 (Ly et al 2017)
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