Molecular basis of human poly(A) polymerase recruitment by mPSF.

3' end processing of most eukaryotic precursor-mRNAs (pre-mRNAs) is a crucial cotranscriptional process that generally involves the cleavage and polyadenylation of the precursor transcripts. Within the human 3' end processing machinery, the four-subunit mammalian polyadenylation specificity factor (mPSF) recognizes the polyadenylation signal (PAS) in the pre-mRNA and recruits the poly(A) polymerase α (PAPOA) to it. To shed light on the molecular mechanisms of PAPOA recruitment to mPSF, we used a combination of cryogenic-electron microscopy (cryo-EM) single-particle analysis, computational structure prediction, and in vitro biochemistry to reveal an intricate interaction network. A short linear motif in the mPSF subunit FIP1 interacts with the structured core of human PAPOA, with a binding mode that is evolutionarily conserved from yeast to human. In higher eukaryotes, however, PAPOA contains a conserved C-terminal motif that can interact intramolecularly with the same residues of the PAPOA structured core used to bind FIP1. Interestingly, using biochemical assay and cryo-EM structural analysis, we found that the PAPOA C-terminal motif can also directly interact with mPSF at the subunit CPSF160. These results show that PAPOA recruitment to mPSF is mediated by two distinct intermolecular connections and further suggest the presence of mutually exclusive interactions in the regulation of 3' end processing.

An examination of the metal ion content in the active sites of human endonucleases CPSF73 and INTS11.

Human cleavage and polyadenylation specificity factor (CPSF)73 (also known as CPSF3) is the endoribonuclease that catalyzes the cleavage reaction for the 3'-end processing of pre-mRNAs. The active site of CPSF73 is located at the interface between a metallo-ß-lactamase domain and a ß-CASP domain. Two metal ions are coordinated by conserved residues, five His and two Asp, in the active site, and they are critical for the nuclease reaction. The metal ions have long been thought to be zinc ions, but their exact identity has not been examined. Here we present evidence from inductively coupled plasma mass spectrometry and X-ray diffraction analyses that a mixture of metal ions, including Fe, Zn, and Mn, is present in the active site of CPSF73. The abundance of the various metal ions is different in samples prepared from different expression hosts. Zinc is present at less than 20% abundance in a sample expressed in insect cells, but the sample is active in cleaving a pre-mRNA substrate in a reconstituted canonical 3'-end processing machinery. Zinc is present at 75% abundance in a sample expressed in human cells, which has comparable endonuclease activity. We also observe a mixture of metal ions in the active site of the CPSF73 homolog INTS11, the endonuclease for Integrator. Taken together, our results provide further insights into the role of metal ions in the activity of CPSF73 and INTS11 for RNA 3'-end processing.

Reconstitution of 3' end processing of mammalian pre-mRNA reveals a central role of RBBP6.

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.

Structural and biochemical analysis of a NOT1 MIF4G-like domain of the CCR4-NOT complex.

The CCR4-NOT complex plays a central role in the regulation of gene expression and degradation of messenger RNAs. The multisubunit complex assembles on the NOT1 protein, which acts as a 'scaffold' and is highly conserved in eukaryotes. NOT1 consists of a series of helical domains that serve as docking sites for other CCR4-NOT subunits. We describe a crystal structure of a connector domain of NOT1 from the thermophilic fungus Chaetomium thermophilum (Ct). Comparative structural analysis indicates that this domain adopts a MIF4G-like fold and we have termed it the MIF4G-C domain. Solution scattering studies indicate that the human MIF4G-C domain likely adopts a very similar fold to the Ct MIF4G-C. MIF4G domains have been described to mediate interactions with DEAD-box helicases such as DDX6. However, comparison of the interfaces of the MIF4G-C with the MIF4G domain of NOT1 that interacts with DDX6 reveals key structural differences that explain why the MIF4G-C does not bind DDX6. We further show that the human MIF4G-C does not interact stably with other subunits of the CCR4-NOT complex. The structural conservation of the MIF4G-C domain suggests that it may have an important but presently undefined role in the CCR4-NOT complex.

GIGYF1/2 proteins use auxiliary sequences to selectively bind to 4EHP and repress target mRNA expression.

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.