2 STRUCTURALLY DIFFERENT RNA MOLECULES ARE BOUND BY THE SPLICEOSOMAL PROTEIN U1A USING THE SAME RECOGNITION STRATEGY

Background: Human U1A protein binds to hairpin II of U1 small nuclear RNA (snRNA) and, together with other proteins, forms the U1 snRNP esse ntial in pre-mRNA splicing, U1A protein also binds to the 3' untransla ted region (S'UTR) of its own pre-mRNA, inhibiting polyadenylation of the 3' end and thereby downregulating its own expression, The 3'UTR fo lds into an evolutionarily conserved secondary structure with two inte rnal loops; one loop contains the sequence AUUGCAC and the other its v ariant AUUGUAC. The sequence AUUGCAC is also found in hairpin II of UI snRNA; hence, U1A protein recognizes the same heptanucleotide sequenc e in two different structural contexts. In order to better understand the control mechanism of the polyadenylation process, we have built a model of the U1A protein-3'UTR complex based on the crystal structure of the U1A protein-hairpin II RNA complex which we determined previous ly, Results: In the crystal structure of the U1A protein-hairpin II RN A complex the AUUGCAC sequence fits tightly into a groove on the surfa ce of U1A protein, The conservation of the heptanucleotide in the 3'UT R strongly suggests that U1A protein forms identical sequence-specific contacts with the heptanucleotide sequence when complexed with the 3' UTR. The crystal structure of the hairpin II complex and the twofold s ymmetry in the 3'UTR RNA provide sufficient information to restrict th e conformation of the 3'UTR RNA and have enabled us to build a model o f the 3'UTR complex. Conclusions: In the U1A-3'UTR complex, sequence-s pecific interactions are made entirely by the conserved heptanucleotid e and the last base pair (C:G) of the stem, The structure is stabilize d by protein-protein contacts and by electrostatic interactions betwee n basic amino acids of the protein and the phosphate backbone of the R NA stem regions. The formation of a protein dimer necessary for the in hibition of poly(A) polymerase requires a conformational change of the C termini of the proteins upon RNA binding. This mechanism could prev ent the inhibition of poly(A) polymerase by free U1A protein, The mode l is consistent with biochemical data, and the protein-protein interac tions within the 3'UTR complex account for the cooperativity of U1A pr otein binding to the 3'UTR. The model also serves as an important stru ctural guide for designing further experiments to understand the inter action between the U1A-3'UTR complex and poly(A) polymerase.