PROBING THE 3'-UTR STRUCTURE OF U1A MESSENGER-RNA AND FOOTPRINTING ANALYSIS OF ITS COMPLEX WITH U1A PROTEIN

The structure of the conserved region of the U1A pre-mRNA (Ag RNA) and its complex with U1A protein was investigated. The previously propose d secondary structure of Ag RNA, derived from enzymatic probing and an alysis of the structure and function of mutant mRNAs, is now confirmed by chemical probing data and further refined in the regions where the enzymatic data were not conclusive. The two unpaired nucleotides in t he internal loops opposite of the Box sequences as well as the tetralo op could not be cleaved by ribonucleases, but are accessible to chemic al probes. Concerning the RNA-protein complex, the protection experime nts showed that the Box regions are largely protected when the U1A pro tein is present. All stem regions in the 5' part of the structure seem protected against ribonucleases. Unexpectedly, the nucleotides of the tetraloop become accessible to ribonucleases in the RNA-protein compl ex. This result indicates that the tetraloop undergoes a conformationa l change upon U1A protein binding. The 3' part of the Ag RNA sequence, containing the polyadenylation signal in a hairpin structure, showed hardly any protection, a finding that agrees with the fact that U1A do es not interfere with the binding of the cleavage polyadenylation spec ificity factor (CPSF) to the polyadenylation signal.