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.