The eIF4E homologous protein (4EHP) is thought to repress translation by competing with eIF4E for binding to the 5 ′ cap structure of specific mRNAs to which it is recruited through interactions with various proteins, including the GRB10-interacting GYF (glycine-tyrosine-phenylalanine domain) proteins 1 and 2 (GIGYF1/2). Despite its similarity to eIF4E, 4EHP does not interact with eIF4G and therefore fails to initiate translation. In contrast to eIF4G, GIGYF1/2 bind selectively to 4EHP but not eIF4E. Here, we present crystal structures of the 4EHP-binding regions of GIGYF1 and GIGYF2 in complex with 4EHP, which reveal the molecular basis for the selectivity of the GIGYF1/2 proteins for 4EHP. Complementation assays in a GIGYF1/2-null cell line using structure-based mutants indicate that 4EHP requires interactions with GIGYF1/2 to down-regulate target mRNA expression. Our studies provide structural insights into the assembly of 4EHP-GIGYF1/2 repressor complexes and reveal that rather than merely facilitating 4EHP recruitment to transcripts, GIGYF1/2 proteins are required for repressive activity.

The 3′ ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction: endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of 14 polypeptides that are essential and two that are stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, cleavage factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, with structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.

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The 3’ ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction, endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of fourteen polypeptides essential and two stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, Cleavage Factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.