SUMMARY We recently reported that two homologous yeast proteins, Rai1 and Dxo1, function in a quality control mechanism to clear cells of incompletely 5′-end capped mRNAs. Here we report that their mammalian homolog, Dom3Z, possesses pyrophosphohydrolase, decapping and 5′-3′ exoribonuclease activities, and will be referred to as DXO. Surprisingly, we find that DXO preferentially degrades defectively capped pre-mRNAs in cells. Further studies show that incompletely capped pre-mRNAs are inefficiently spliced at all introns, in contrast to current understanding, and poorly cleaved for polyadenylation. Crystal structures of DXO in complex with substrate mimic and products at up to 1.5Å resolution provide elegant insights into the catalytic mechanism and molecular basis for its three apparently distinct activities. Our data reveal a pre-mRNA 5′-end capping quality control mechanism in mammalian cells, with DXO as the central player for this mechanism, and demonstrate an unexpected intimate link between proper 5′-end capping and subsequent pre-mRNA processing.

Summary Recent studies showed that Rai1 is a crucial component of the mRNA 5′-end capping quality control mechanism in yeast. The yeast genome encodes a weak homolog of Rai1, Ydr370C, but little is known about this protein. Here we report the crystal structures of Kluyveromyces lactis Ydr370C and the first biochemical and functional studies on this protein. The overall structure of Ydr370C is similar to Rai1. Ydr370C has robust decapping activity on RNAs with unmethylated caps but it has no detectable pyrophosphohydrolase activity. Unexpectedly, Ydr370C also possesses distributive, 5′-3′ exoribonuclease activity, and we propose the name Dxo1 for this novel eukaryotic enzyme with both decapping and exonuclease activities. Studies in yeast where both Dxo1 and Rai1 are disrupted reveal that mRNAs with incomplete caps are produced even under normal growth conditions, in sharp contrast to current understanding of the capping process.

Repression of E2F transcription activity by the retinoblastoma (Rb) tumor suppressor through its interaction with the transactivation domain of the E2F transcription factor is one of the central features of G1/S arrest in the mammalian cell cycle. Deregulation of the Rb-E2F interaction results in hyperproliferation, lack of differentiation, and apoptosis, and can lead to cancer. The 2.2-Å crystal structure of the Rb pocket complexed with an 18-residue transactivation-domain peptide of E2F-2 reveals that the boomerang-shaped peptide binds to the highly conserved interface between the A-box and the B-box of the Rb pocket in a bipartite manner. The N-terminal segment of the E2F-2 peptide in an extended ␤-strand-like structure interacts with helices from the conserved groove at the A-B interface, whereas the C-terminal segment, which contains one 3 10 helix, binds to a groove mainly formed by A-box helices. The flexibility in the middle of the E2F-2 peptide is essential for the tight association of E2F to the Rb pocket. The binding of Rb to the E2F-2 peptide conceals several conserved residues that are crucial for transcription activation of E2F. We provide the structural basis for the Rb-mediated repression of E2F transcription activity without the requirement of histone-modifying enzymes.

The methylation of lysine residues of histones plays a pivotal role in the regulation of chromatin structure and gene expression. Here, we report two crystal structures of SET7/9, a histone methyltransferase (HMTase) that transfers methyl groups to Lys4 of histone H3, in complex with S-adenosyl-L-methionine (AdoMet) determined at 1.7 and 2.3 A Ê resolution. The structures reveal an active site consisting of: (i) a binding pocket between the SET domain and a c-SET helix where an AdoMet molecule in an unusual conformation binds; (ii) a narrow substrate-speci®c channel that only unmethylated lysine residues can access; and (iii) a catalytic tyrosine residue. The methyl group of AdoMet is directed to the narrow channel where a substrate lysine enters from the opposite side. We demonstrate that SET7/9 can transfer two but not three methyl groups to unmodi®ed Lys4 of H3 without substrate dissociation. The unusual features of the SET domain-containing HMTase discriminate between the un-and methylated lysine substrate, and the methylation sites for the histone H3 tail. Keywords: compact form of AdoMet/9 histone methyltransferase/post-SET helix/SET7/SET domain/ substrate-speci®c channel

The NAD-dependent histone/protein deacetylase activity of Sir2 (silent information regulator 2) accounts for its diverse biological roles including gene silencing, DNA damage repair, cell cycle regulation, and life span extension. We provide crystallographic evidence that 2-O-acetyl ADP-ribose is the reaction product that is formed at the active site of Sir2 from the 2.6-Å co-crystal structure of 2-O-acetyl-ADP-ribose and Sir2 from Archaeoglobus fulgidus. In addition, we show that His-116 and Phe-159 play critical roles in the catalysis and substrate recognition. The conserved Ser-24 and Asp-101 contribute to the stability for NAD binding rather than being directly involved in the catalysis. The crystal structures of wild type and mutant derivatives of Sir2, in conjunction with biochemical analyses of the mutants, provide novel insights into the reaction mechanism of Sir2-mediated deacetylation.

The 5′→ 3′ exoribonucleases (XRNs) have important functions in transcription, RNA metabolism, and RNA interference. The recent structure of Rat1 (Xrn2) showed that the two highly conserved regions of XRNs form a single, large domain, defining the active site of the enzyme. Xrn1 has a 510-residue segment following the conserved regions that is required for activity but is absent in Rat1. We report here the crystal structures at 2.9 Å resolution of Kluyveromyces lactis Xrn1 (residues 1–1245, E178Q mutant), alone and in complex with a Mn2+ ion in the active site. The 510-residue segment contains four domains (D1–D4), located far from the active site. Our mutagenesis and biochemical studies demonstrate that their functional importance is due to their stabilization of the conformation of the N-terminal segment of Xrn1. These domains may also constitute a platform for interacting with protein partners of Xrn1.

The Mus81-Eme1 complex is a structure-specific endonuclease that plays an important role in rescuing stalled replication forks and resolving the meiotic recombination intermediates in eukaryotes. We have determined the crystal structure of the Mus81-Eme1 complex. Both Mus81 and Eme1 consist of a central nuclease domain, two repeats of the helix-hairpin-helix (HhH) motif at their C-terminal region, and a linker helix. While each domain structure resembles archaeal XPF homologs, the overall structure is significantly different from those due to the structure of a linker helix. We show that a flexible intradomain linker that formed with 36 residues in the nuclease domain of Eme1 is essential for the recognition of DNA. We identified several basic residues lining the outer surface of the active site cleft of Mus81 that are involved in the interaction with a flexible arm of a nicked Holliday junction (HJ). These interactions might contribute to the optimal positioning of the opposite junction across the nick into the catalytic site, which provided the basis for the "nick and counternick" mechanism of Mus81-Eme1 and for the nicked HJ to be the favored in vitro substrate of this enzyme.[Keywords: Structure-specific endonuclease; nicked Holliday junction; substrate preference; Mus81-Eme1; crystal structure] Supplemental material is available at http://www.genesdev.org.

Tumor suppressor programmed cell death protein 4 (PDCD4) inhibits the translation initiation factor eIF4A, an RNA helicase that catalyzes the unwinding of secondary structure at the 5 -untranslated region of mRNAs and controls the initiation of translation. Here, we determined the crystal structure of the human eIF4A and PDCD4 complex. The structure reveals that one molecule of PDCD4 binds to the two eIF4A molecules through the two different binding modes. While the two MA3 domains of PDCD4 bind to one eIF4A molecule, the C-terminal MA3 domain alone of the same PDCD4 also interacts with another eIF4A molecule. The eIF4A-PDCD4 complex structure suggests that the MA3 domain(s) of PDCD4 binds perpendicular to the interface of the two domains of eIF4A, preventing the domain closure of eIF4A and blocking the binding of RNA to eIF4A, both of which are required events in the function of eIF4A helicase. The structure, together with biochemical analyses, reveals insights into the inhibition mechanism of eIF4A by PDCD4 and provides a framework for designing chemicals that target eIF4A.translation inhibition ͉ tumor suppressor ͉ RNA helicase ͉ domain closure ͉ MA3 domain P rogrammed cell death protein 4 (PDCD4) is a translation inhibitor that suppresses neoplastic transformation in cultured cells and transgenic mice (1-3). Loss or reduced expression of PDCD4 has been implicated in the development and progression of a variety of aggressive human cancers (4-6). PDCD4 is regulated by the S6K1 kinase and the SCF ␤TRCP ubiquitin ligase in response to the activation of the mTOR pathway by mitogens, and the controlled degradation of PDCD4 is essential for efficient protein synthesis and consequently for cell growth and proliferation (7).PDCD4 is believed to perform its tumor suppressor function primarily through interaction with eIF4A and eIF4G, which are components of mRNA-binding complex eIF4F (8). eIF4A is a DEAD-box RNA helicase, with two domains, that unwinds the secondary structures in 5Ј-untranslated region (UTR) and cap of mRNA and thereby facilitates ribosome scanning (8). eIF4G is an adaptor protein that coordinates assembly of translation factors and the small ribosomal subunit (8). PDCD4 is believed to inhibit cap-dependent translation by directly inhibiting the helicase activity of eIF4A or by competing with eIF4G for binding to eIF4A and preventing assembly into a eukaryotic initiation complex, eIF4F, or both (1, 9, 10).PDCD4 is formed with the two MA3 domains at its middle (mMA3) and C-terminal regions (cMA3) (9, 11-13). Mutational and NMR binding analysis have shown that PDCD4 uses both MA3 domains to interact with eIF4A and prevents translation (9, 10). However, other studies have demonstrated that the cMA3 domain alone is sufficient for the inhibition of RNA helicase and translation (12). The interactions between eIF4A and PDCD4 have been analyzed in several mutational and NMR mapping studies (1, 9, 10). Nevertheless, it is unclear from these studies how PDCD4 inhibits eIF4A at the molecular level. To elucidate ...