Visualization of the interaction of a regulatory protein with RNA in vivo

We have adapted to RNA molecules the ligation-mediated polymerase chain reaction (LMPCR) procedure of genomic sequencing [Mueller, P. R. & Wold, B. (1989) Science 246, 780-786]. This new procedure, the reverse ligation-mediated PCR (RLPCR), is sufficiently sensitive to allow "in vivo" footprinting of minor RNA species. It is based on the ligation of an RNA linker of known sequence to every 5' end resulting from the cleavage of total cellular RNA. Target RNA molecules are specifically reverse-transcribed and the resulting products are amplified by PCR. The localization of the initial 5' ends is ultimately determined on a sequencing gel. To demonstrate the validity of this strategy, we have used RNase T1 treatment of permeabilized cells and RLPCR and have detected in vivo iron-depletion-dependent footprints on two iron-responsive elements of the transferrin receptor mRNA.