Processing mRNA 3' Ends In Vitro

The 3′ ends of most nonhistone mRNAs in mammalian cells are generated by the endonucleolytic cleavage of an mRNA precursor (pre-mRNA) followed by the addition of a polyadenylate (poly[A]) tail (see ref. 1 for a recent review on mRNA 3′ end processing). The ability to process pre-mRNAs in vitro has contributed greatly to our understanding of the biochemical details of the reaction and, more recently, the regulation of this processing event. The in vitro conditions are such that the same cis -acting signals on the pre-mRNA that are required for processing in vivo are also required in vitro. The assay can be performed in one of two ways. In the first, a plasmid encoding a gene containing the poly(A) site of interest is incubated in a cell-free extract under conditions that allow transcription of the gene by RNA polymerase II and processing of the nascent transcript (2 ). In the second, the pre-mRNA is synthesized in a separate reaction using standard bacteriophage polymerase-driven systems, and then incubated in the cell-free extract (3 ). These are referred to as transcription- processing and processing reactions, respectively. The reaction conditions can be adjusted so as to examine only cleavage of the precursor, to examine cleavage and addition of the poly(A) tail, or to examine the addition of a poly(A) tail to a precleaved RNA molecule. The products of the reaction are separated from the precursor by standard denaturing gel electrophoresis, allowing for detection using autoradiographic techniques.