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...

Ezh2 functions as a histone H3 Lys 27 (H3K27) methyltransferase when comprising the Polycomb-Repressive Complex 2 (PRC2). Trimethylation of H3K27 (H3K27me3) correlates with transcriptionally repressed chromatin. The means by which PRC2 targets specific chromatin regions is currently unclear, but noncoding RNAs (ncRNAs) have been shown to interact with PRC2 and may facilitate its recruitment to some target genes. Here we show that Ezh2 interacts with HOTAIR and Xist. Ezh2 is phosphorylated by cyclin-dependent kinase 1 (CDK1) at threonine residues 345 and 487 in a cell cycle-dependent manner. A phospho-mimic at residue 345 increased HOTAIR ncRNA binding to Ezh2, while the phospho-mimic at residue 487 was ineffectual. An Ezh2 domain comprising T345 was found to be important for binding to HOTAIR and the 5′ end of Xist.

Summary JARID2 is an accessory component of Polycomb repressive complex-2 (PRC2) required for the differentiation of embryonic stem cells (ESCs). A role for JARID2 in the recruitment of PRC2 to target genes silenced during differentiation has been put forward, but the molecular details remain unclear. We identified a 30-amino acid region of JARID2 that mediates interactions with long noncoding RNAs (lncRNAs) and found that the presence of lncRNAs stimulated JARID2–EZH2 interactions in vitro and JARID2-mediated recruitment of PRC2 to chromatin in vivo. Native and crosslinked RNA immunoprecipitations of JARID2 revealed that Meg3 and other lncRNAs from the imprinted Dlk1-Dio3 locus, an important regulator of development, interacted with PRC2 via JARID2. Lack of MEG3 expression in human induced pluripotent cells altered the chromatin distribution of JARID2, PRC2, and H3K27me3. Our findings show that lncRNAs facilitate JARID2–PRC2 interactions on chromatin and suggest a mechanism by which lncRNAs contribute to PRC2 recruitment.

EZH2 is the catalytic subunit of PRC2, a central epigenetic repressor essential for development processes in vivo and for the differentiation of embryonic stem cells (ESCs) in vitro. The biochemical function of PRC2 in depositing repressive H3K27me3 marks is well understood, but how it is regulated and directed to specific genes before and during differentiation remains unknown. Here, we report that PRC2 binds at low levels to a majority of promoters in mouse ESCs, including many that are active and devoid of H3K27me3. Using in vivo RNA–protein crosslinking, we show that EZH2 directly binds to the 5′ of nascent RNAs transcribed from a subset of these promoters and that these binding events correlate with decreased H3K27me3. Our findings suggest a molecular mechanism by which PRC2 senses the transcriptional state of the cell and translates it into epigenetic information.