Most eukaryotic messenger RNA precursors (pre-mRNAs) undergo extensive maturational processing, including 3'-end cleavage and polyadenylation [1][2][3][4][5][6][7][8] . Despite the characterization of a large number of proteins that are required for the cleavage reaction, the identity of the endoribonuclease is not known 4,9,10 . Recent analyses suggested that the 73 kD subunit of cleavage and polyadenylation specificity factor (CPSF-73) may be the endonuclease for this and related reactions [10][11][12][13][14][15] , although no direct data confirmed this. Here we report the crystal structures of human CPSF-73 at 2.1 Å resolution, complexed with zinc ions and a sulfate that may mimic the phosphate group of the substrate, and the related yeast protein CPSF-100 (Ydh1p) at 2.5 Å resolution. Both CPSF-73 and CPSF-100 contain two domains, a metallo-β-lactamase domain and a novel β-CASP domain. The active site of CPSF-73, with two zinc ions, is located at the interface of the two domains. Purified recombinant CPSF-73 possesses endoribonuclease activity, and mutations that disrupt zinc binding in the active site abolish this activity. Our studies provide the first direct experimental evidence that CPSF-73 is the pre-mRNA 3'-end processing endonuclease. Keywordspolyadenylation; metallo-β-lactamase; pre-mRNA processing; Artemis; V(D)J recombination; double-strand break repair CPSF-73 belongs to the metallo-β-lactamase superfamily of zinc-dependent hydrolases 11,12 . Canonical metallo-β-lactamases contain five signature sequence motifs-Asp (motif 1), His-X-His-X-Asp-His (motif 2), His (motif 3), Asp (motif 4) and His (motif 5), most of which are ligands to the two zinc ions in their active site. Sequence conservation between CPSF-73 and the canonical metallo-β-lactamases is limited to these signature motifs. While the first four motifs can be identified in the N-terminal segment of CPSF-73 (Supplemental Fig. 1a, Supplemental Table 1), the fifth motif was uncertain, with three candidates, A (Asp or Glu), B (His), and C (His) (Supplemental Fig. 1a), in the so-called β-CASP motif 12 . Motif B was proposed to be equivalent to motif 5 in the canonical metallo-β-lactamases. Another subunit of CPSF, CPSF-100, shares sequence conservation (Supplemental Fig. 1b) Fig. 1a) with CPSF-73 but lacks the putative Zn 2+ binding residues.To understand the roles of CPSF-73 and CPSF-100 in pre-mRNA 3'-end processing, we determined the structures of human CPSF-73 (residues 1-460), and yeast CPSF-100 (residues 1-720) (the crystallographic data are summarized in Supplemental Table 2). The two structures obtained for CPSF-73 were crystallized in the absence or presence of 0.5 mM zinc (although both structures contained zinc atoms; see below). We discovered serendipitously that in situ proteolysis by a fungal protease is crucial for the crystallization of yeast CPSF-100 16 .The structure of CPSF-73 can be divided into two domains (Fig. 1a). The N-terminal residues (amino acids 1-208) form a domain similar to the structure of canonical me...

High-voltage-activated Ca2+ channels are essential for diverse biological processes. They are composed of four or five subunits, including alpha1, alpha2-delta, beta and gamma (ref. 1). Their expression and function are critically dependent on the beta-subunit, which transports alpha1 to the surface membrane and regulates diverse channel properties. It is believed that the beta-subunit interacts with alpha1 primarily through the beta-interaction domain (BID), which binds directly to the alpha-interaction domain (AID) of alpha1; however, the molecular mechanism of the alpha1-beta interaction is largely unclear. Here we report the crystal structures of the conserved core region of beta3, alone and in complex with AID, and of beta4 alone. The structures show that the beta-subunit core contains two interacting domains: a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain. The AID binds to a hydrophobic groove in the GK domain through extensive interactions, conferring extremely high affinity between alpha1 and beta-subunits. The BID is essential both for the structural integrity of and for bridging the SH3 and GK domains, but it does not participate directly in binding alpha1. The presence of multiple protein-interacting modules in the beta-subunit opens a new dimension to its function as a multi-functional protein.

Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12, and plays pivotal roles in transcriptional regulation. The catalytic subunit EZH2 methylates histone H3 lysine 27 (H3K27), and its activity is further enhanced by the binding of EED to trimethylated H3K27 (H3K27me3). Small-molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported. Here we report the discovery of EED226, a potent and selective PRC2 inhibitor that directly binds to the H3K27me3 binding pocket of EED. EED226 induces a conformational change upon binding EED, leading to loss of PRC2 activity. EED226 shows similar activity to SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing a mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show that EED226 inhibits PRC2 activity via an allosteric mechanism and offers an opportunity for treatment of PRC2-dependent cancers.

BackgroundA high-throughput genotyping platform is needed to enable marker-assisted breeding in the allo-octoploid cultivated strawberry Fragaria × ananassa. Short-read sequences from one diploid and 19 octoploid accessions were aligned to the diploid Fragaria vesca ‘Hawaii 4’ reference genome to identify single nucleotide polymorphisms (SNPs) and indels for incorporation into a 90 K Affymetrix® Axiom® array. We report the development and preliminary evaluation of this array.ResultsAbout 36 million sequence variants were identified in a 19 member, octoploid germplasm panel. Strategies and filtering pipelines were developed to identify and incorporate markers of several types: di-allelic SNPs (66.6%), multi-allelic SNPs (1.8%), indels (10.1%), and ploidy-reducing “haploSNPs” (11.7%). The remaining SNPs included those discovered in the diploid progenitor F. iinumae (3.9%), and speculative “codon-based” SNPs (5.9%). In genotyping 306 octoploid accessions, SNPs were assigned to six classes with Affymetrix’s “SNPolisher” R package. The highest quality classes, PolyHigh Resolution (PHR), No Minor Homozygote (NMH), and Off-Target Variant (OTV) comprised 25%, 38%, and 1% of array markers, respectively. These markers were suitable for genetic studies as demonstrated in the full-sib family ‘Holiday’ × ‘Korona’ with the generation of a genetic linkage map consisting of 6,594 PHR SNPs evenly distributed across 28 chromosomes with an average density of approximately one marker per 0.5 cM, thus exceeding our goal of one marker per cM.ConclusionsThe Affymetrix IStraw90 Axiom array is the first high-throughput genotyping platform for cultivated strawberry and is commercially available to the worldwide scientific community. The array’s high success rate is likely driven by the presence of naturally occurring variation in ploidy level within the nominally octoploid genome, and by effectiveness of the employed array design and ploidy-reducing strategies. This array enables genetic analyses including generation of high-density linkage maps, identification of quantitative trait loci for economically important traits, and genome-wide association studies, thus providing a basis for marker-assisted breeding in this high value crop.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1310-1) contains supplementary material, which is available to authorized users.