hnRNP A1 binds promiscuously to oligoribonucleotides: utilization of random and homo-oligonucleotides to discriminate sequence from base-specific binding

Abstract

To understand the range of possible and probable A1 functions in pre-mRNA biogenesis, it is important that we quantify the relative ability (or inability) of A1 to bind high affinity RNA target sequences and/or structures. Using a fluorescence competition assay we have determined apparent binding affinities for a wide range of 20mer oligos containing putative and possible A1 targets including the high affinity 'winner' sequence identified by selection/amplification [Burd,C.G and Dreyfuss,G. (1994) EMBO J. 13, 1197-1204], AUUUA sequences found in 3'-UTRs of labile mRNAs, 5'- and 3'-splice sites and telomeric sequences. With the exception of a 20mer 'winner' sequence, all other 20mers examined bind A1 with a narrow, approximately 10-fold range of affinities extending from 3.2 x 10(6) to 4.2 x 10(7) M(-1). Studies with homo-oligomers suggest this range reflects nucleotide base rather than sequence specificity and hence, it was possible to predict reasonably accurate affinities for all other 20mers examined except for the 'winner', whose unusually high affinity of 4.0 x 10(8) M(-1) results from a unique higher order structure and sequence. Since there is no known physiological role for the 'winner' 20mer sequence, these data suggest A1 generally binds indiscriminately to all available pre-mRNA sequences. Both the large abundance of A1 in vivo and its binding properties are thus consistent with it playing a structural role in pre-mRNA biogenesis.