Probing the 3‘ UTR Structure of U1A mRNA and Footprinting Analysis of Its Complex with U1A Protein

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Abstract
The structure of the conserved region of the U1A pre-mRNA (Ag RNA) and its complex with U1A protein was investigated. The previously proposed secondary structure of Ag RNA, derived from enzymatic probing and analysis 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 the internal loops opposite of the Box sequences as well as the tetraloop could not be cleaved by ribonucleases, but are accessible to chemical probes. Concerning the RNA−protein complex, the protection experiments showed that the Box regions are largely protected when the U1A protein 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 complex. This result indicates that the tetraloop undergoes a conformational 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 does not interfere with the binding of the cleavage polyadenylation specificity factor (CPSF) to the polyadenylation signal.