title: Oligo(dT)-primed RT-PCR isolation of polyadenylated RNA degradation intermediates.
abstract: The posttranscriptional modification of RNA by polyadenylation serves various purposes, among them to assist in RNA degradation (see an alternative protocol for measuring RNA degradation on Method for measuring mRNA decay rate in Saccharomyces cerevisiae). This function, once thought to occur in prokaryotic or organellar systems alone, is now known to operate in the nuclei and cytoplasm of eukaryotes as well (Slomovic et al., 2008; Slomovic et al., 2010; Houseley and Tollervey, 2009; Deutscher, 2006). Poly(A)-assisted RNA decay begins with the endonucleolytic cleavage of the transcript. Following this, a poly(A) or oligo(A) tail is added to the 3' end of the cleavage product. This tag serves as a 'landing pad' for 3'-5' exoribonucleases that then begin to digest the RNA fragment. Truncated RNA molecules that have undergone tail addition but have yet to be degraded are called degradation intermediates. The detection of such intermediates is considered a tell-tale sign that poly(A)-assisted RNA decay occurs in the organism being studied. Determination of the tail nucleotide composition by DNA sequencing often aids the researcher in identifying the enzyme responsible for tail synthesis since tails can be either homopolymeric (exclusively A residues) or heteropolymeric (A-rich tails that may include other nucleotides). The following protocol, based on oligo(dT)-primed reverse transcription, describes the step-by-step detection and isolation of adenylated degradation intermediates in the study of poly(A)-assisted RNA decay.
authors.affiliation: Biology Faculty, Technion Institute of Technology, Haifa, Israel.
authors.forename: Shimyn
authors.initials: S
authors.lastname: Slomovic
authors.forename: Gadi
authors.initials: G
authors.lastname: Schuster
mesh_terms: D018274:Electroporation / Q000379:methods; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D012333:RNA, Messenger
keywords: Colony screening; Electroporation and plating; Gel purification; Oligo(dT)-primed reverse transcription; Plasmid DNA isolation and sequencing; Polymerase chain reaction (PCR)