Transcriptome-wide RNA processing kinetics revealed using extremely short 4tU labeling

J David Barrass, Jane E A Reid, Yuanhua Huang, Ralph D Hector, Guido Sanguinetti, Jean D Beggs, Sander Granneman

Research output: Contribution to journalArticlepeer-review

Abstract

BACKGROUND: RNA levels detected at steady state are the consequence of multiple dynamic processes within the cell. In addition to synthesis and decay, transcripts undergo processing. Metabolic tagging with a nucleotide analog is one way of determining the relative contributions of synthesis, decay and conversion processes globally.

RESULTS: By improving 4-thiouracil labeling of RNA in Saccharomyces cerevisiae we were able to isolate RNA produced during as little as 1 minute, allowing the detection of nascent pervasive transcription. Nascent RNA labeled for 1.5, 2.5 or 5 minutes was isolated and analyzed by reverse transcriptase-quantitative polymerase chain reaction and RNA sequencing. High kinetic resolution enabled detection and analysis of short-lived non-coding RNAs as well as intron-containing pre-mRNAs in wild-type yeast. From these data we measured the relative stability of pre-mRNA species with different high turnover rates and investigated potential correlations with sequence features.

CONCLUSIONS: Our analysis of non-coding RNAs reveals a highly significant association between non-coding RNA stability, transcript length and predicted secondary structure. Our quantitative analysis of the kinetics of pre-mRNA splicing in yeast reveals that ribosomal protein transcripts are more efficiently spliced if they contain intron secondary structures that are predicted to be less stable. These data, in combination with previous results, indicate that there is an optimal range of stability of intron secondary structures that allows for rapid splicing.

Original languageEnglish
Pages (from-to)282-
JournalGenome Biology
Volume16
Issue number1
DOIs
Publication statusPublished - 17 Dec 2015

Keywords

  • cryptic transcripts
  • metabolic labeling
  • RNA sequencing
  • splicing
  • yeast

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