Simultaneous Measurement of Transcriptional and Post-transcriptional Parameters by 3' End RNA-Seq

Cellular RNA levels are determined by transcription and decay rates, which are fundamental in understanding gene expression regulation. Measurement of these two parameters is usually performed independently, complicating analysis as well as introducing methodological biases and batch effects that hamper direct comparison. Here, we present a simple approach of concurrent sequencing of S. cerevisiae poly(A)⁺ and poly(A)^- RNA 3' ends to simultaneously estimate total RNA levels, transcription, and decay rates from the same RNA sample. The transcription data generated correlate well with reported estimates and also reveal local RNA polymerase stalling and termination sites with high precision. Although the method by design uses brief metabolic labeling of newly synthesized RNA with 4-thiouracil, the results demonstrate that transcription estimates can also be gained from unlabeled RNA samples. These findings underscore the potential of the approach, which should be generally applicable to study a range of biological questions in diverse organisms.