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

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Abstract
The cleavage factor Im (CF Im), consists of a 25 kDa subunit (CF Im25) and one of three larger subunits (CF Im59, CF Im68, CF Im72), 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 Im, comprising CF Im25 and the RNA recognition motif domain of CF Im68 (CF Im68RRM), and the crystal structure of the CF Im-RNA complex. These structures show that two CF Im68RRM molecules bind to the CF Im25 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 Im25-CF Im68RRM heterotetramer, providing structural basis for the mechanism by which CF Im binds two UGUAA elements within one molecule of pre-mRNA simultaneously. Point mutation and kinetic analyses demonstrate that CF Im68RRM can bind the immediately flanking upstream region of the UGUAA element, and CF Im68RRM binding significantly increases the RNA-binding affinity of the complex, suggesting that CF Im68 makes an essential contribution to pre-mRNA binding.

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