Detection and characterization of polyadenylated RNA in Eukarya, Bacteria, Archaea, and organelles

The posttranscriptional addition of poly(A) extensions to RNA is a phenomenon common to almost all organisms. In eukaryotes, a stable poly(A) tail is added to the 3'-end of most nucleus-encoded mRNAs, as well as to mitochondrion-encoded transcripts in animal cells. In prokaryotes and organelles, RNA molecules are polyadenylated as part of a polyadenylation-stimulated RNA degradation pathway. In addition, polyadenylation of nucleus-encoded transcripts in yeast and human cells was recently reported to promote RNA degradation. Not only homopolymeric poly(A) tails, composed exclusively of adenosines, but also heteropolymeric poly(A)-rich extensions, which include the other three nucleotides as well, have been observed in bacteria, archaea, chloroplasts, and human cells. In most instances, the detection of nonabundant truncated transcripts with posttranscriptionally added poly(A) or poly(A)-rich extensions serves as a telltale sign of the presence of a polyadenylation-stimulated RNA degradation pathway. In this chapter, we describe several methods found to be efficient in detecting and characterizing polyadenylated transcripts in bacteria, archaea, organelles, and nucleus-encoded RNAs. Detailed protocols for the oligo(dT)- and circularized reverse transcription (cRT) PCR methods, as well as the ribonuclease digestion method, are outlined, along with examples of results obtained with these techniques.