Structural basis of pre-mRNA recognition by the human cleavage factor Im complex

The cleavage factor I-m (CF I-m), consists of a 25 kDa subunit (CF I(m)25) and one of three larger subunits (CF I(m)59, CF I(m)68, CF I(m)72), and is an essential protein complex for pre-mRNA 3'-end cleavage and polyadenylation. It recognizes the upstream sequence of the poly(A) site in a sequence-dependent manner. Here we report the crystal structure of human CF I-m, comprising CF I(m)25 and the RNA recognition motif domain of CF I(m)68 (CF I(m)68RRM), and the crystal structure of the CF I-m-RNA complex. These structures show that two CF I(m)68RRM molecules bind to the CF I(m)25 dimer via a novel RRM-protein interaction mode forming a heterotetramer. The RNA-bound structure shows that two UGUAA RNA sequences, with anti-parallel orientation, bind to one CF I(m)25-CF I(m)68RRM heterotetramer, providing structural basis for the mechanism by which CF I-m binds two UGUAA elements within one molecule of pre-mRNA simultaneously. Point mutation and kinetic analyses demonstrate that CF I(m)68RRM can bind the immediately flanking upstream region of the UGUAA element, and CF I(m)68RRM binding significantly increases the RNA-binding affinity of the complex, suggesting that CF I(m)68 makes an essential contribution to pre-mRNA binding.