Abstract
The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.
Highlights
RNA has a dynamic nature; soon after it is produced much RNA is rapidly processed and degraded
Typical yields for nsRNA recovered using this extremely rapid and specific 4 thiouracil protocol (ers4tU) protocol are displayed in Figure 1b, this has been produced by a bioanalyzer and the trace shows yield of RNA versus size
After about 45 s to 1 min, the amounts of lariats and premRNA reach equilibrium with as much of these RNA species being created by transcription as are processed away by splicing
Summary
RNA has a dynamic nature; soon after it is produced much RNA is rapidly processed and degraded. The concentration (10 μM) used here, and the extremely short labelling times, minimize deleterious effects[5] (Figure 1a), while still yielding sufficient RNA for analysis This technique can be combined with rapid and specific auxin-mediated depletion of a target protein[6,7] (Figure 2), referred to as the "β-est AID 4U" protocol, in which β-estradiol regulated expression of the auxin inducible degron (AID) system is combined with 4tU labelling. In continuous labelling the 4tU is added to the culture and samples taken at regular intervals This type of experiment shows how the RNA is processed and how levels change over time. This type of experiment is useful for monitoring the effect of a metabolic change on RNA processing (see Figure 3d). See Barrass et al.[7] for the detailed AID depletion protocol
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