Nuclear actin and actin-related proteins (Arps) are integral components of various chromatin-remodelling complexes. Actin in such nuclear assemblies does not form filaments but associates in defined complexes, for instance with Arp4 and Arp8 in the INO80 remodeller. To understand the relationship between nuclear actin and its associated Arps and to test the possibility that Arp4 and Arp8 help maintain actin in defined states, we structurally analysed Arp4 and Arp8 from Saccharomyces cerevisiae and tested their biochemical effects on actin assembly and disassembly. The solution structures of isolated Arp4 and Arp8 indicate them to be monomeric and the crystal structure of ATP-Arp4 reveals several differences to actin that explain why Arp4 does not form filaments itself. Remarkably, Arp4, assisted by Arp8, influences actin polymerization in vitro and is able to depolymerize actin filaments. Arp4 likely forms a complex with monomeric actin via the barbed end. Our data thus help explaining how nuclear actin is held in a discrete complex within the INO80 chromatin remodeller.

Poly(C)-binding proteins (PCBPs) are KH (hnRNP K homology) domain-containing proteins that recognize poly(C) DNA and RNA sequences in mammalian cells. Binding poly(C) sequences via the KH domains is critical for PCBP functions. To reveal the mechanisms of KH domain-D/RNA recognition and its functional importance, we have determined the crystal structures of PCBP2 KH1 domain in complex with a 12-nucleotide DNA corresponding to two repeats of the human C-rich strand telomeric DNA and its RNA equivalent. The crystal structures reveal molecular details for not only KH1-DNA/RNA interaction but also protein-protein interaction between two KH1 domains. NMR studies on a protein construct containing two KH domains (KH1 + KH2) of PCBP2 indicate that KH1 interacts with KH2 in a way similar to the KH1-KH1 interaction. The crystal structures and NMR data suggest possible ways by which binding certain nucleic acid targets containing tandem poly(C) motifs may induce structural rearrangement of the KH domains in PCBPs; such structural rearrangement may be crucial for some PCBP functions.

Poly(C)-binding proteins (PCBPs) 2 are KH (hnRNP-K-homology) domain-containing proteins that specifically recognize poly(C) D/RNA sequences (1, 2). There are five PCBPs in mammalian cells: PCBP1-4 and hnRNP K. Each PCBP contains three KH domains: two consecutive domains at the N terminus and a third domain at the C terminus; an intervening sequence of variable length is present between the second and third domains (Fig. 1A).PCBPs regulate gene expression at various levels, including transcription, mRNA processing, mRNA stabilization, and translation, among others. For example, specific binding of hnRNP K and PCBP1 to the single-stranded pyrimidine-rich promoter sequence of the human c-myc gene and mu-opioid receptor (MOR) gene, respectively, activates transcription (3, 4).Binding of PCBP1 or PCBP2 to cellular mRNAs harboring tandem poly(C) motifs within the 3Ј-UTRs stabilize these mRNAs, including ␣-globin, ␤-globin, collagen-␣1, tyrosine hydroxylase, erythropoietin, rennin, and hTERT mRNAs (5-13). In the case of ␣-globin mRNA, it was established that the stoichiometry of the RNA-protein complex (the ␣-complex) is 1:1, and a minimum RNA sequence of 20-nt (5Ј-CCCAACGGGCCCUCCUCCCC-3Ј) is necessary and sufficient for forming the complex (14).Interaction of two PCBPs, hnRNP K, and PCBP1/2, with a multiply tandem C-rich sequence (differentiation control element, DICE.) within the 3Ј-UTR of 15-lipoxygenase (LOX) mRNA leads to translational silencing of the mRNA in erythroid precursor cells (15-17). DICE contains 10 gapless C-rich repeats. The sequence for one repeat is 5Ј-CCCCACCCUCU-UCCCCAAG-3Ј. A minimum of two repeats is required for efficient translational suppression.PCBPs can also activate translation of cellular mRNAs. For example, binding of PCBP1 to an 18-nt C-rich sequence (5Ј-CUCCAUUCCCACUCCCU-3Ј) within the 5Ј-UTR of folate receptor mRNA up-regulates its translation (18). Binding of PCBP1/2 to the acute box cis-element in human heavy ferritin mRNA 5Ј-UTR also enhances translation (19).Besides cellular mRNAs, PCBPs also participate in regulating critical viral RNA functions. Binding of PCBP1/2 to two cisacting C-rich sequence-containing RNA elements within the 5Ј-UTR of poliovirus mRNA (also the genomic RNA) is critical for regulation of cap-independent translation and replication of the viral RNA (20 -24).The mechanistic details are not well understood for any of the PCBP functions. What emerges as a common feature is the binding of PCBPs to C-rich sequence motifs (often present in tandem) of the target D/RNAs. The molecular basis of PCBP KH domains-D/RNA interactions has been revealed by a number of crystal structures of individual KH1 or KH3 domain from PCBPs in complex with C-rich D/RNA sequences (25-28). However, there are no structures with KH2.Little is known about the events subsequent to any KH domain-D/RNA interaction. Pertinent to this point, there are no structures of PCBP constructs containing more than one KH * This work was supported, in whole or in part, by National Institutes of Health Gr...

Nuclear actin-related proteins (Arps) are subunits of several chromatin remodelers, but their molecular functions within these complexes are unclear. We report the crystal structure of the INO80 complex subunit Arp8 in its ATP-bound form. Human Arp8 has several insertions in the conserved actin fold that explain its inability to polymerize. Most remarkably, one insertion wraps over the active site cleft and appears to rigidify the domain architecture, while active site features shared with actin suggest an allosterically controlled ATPase activity. Quantitative binding studies with nucleosomes and histone complexes reveal that Arp8 and the Arp8–Arp4–actin-HSA sub-complex of INO80 strongly prefer nucleosomes and H3–H4 tetramers over H2A–H2B dimers, suggesting that Arp8 functions as a nucleosome recognition module. In contrast, Arp4 prefers free (H3–H4)2 over nucleosomes and may serve remodelers through binding to (dis)assembly intermediates in the remodeling reaction.

KH (hnRNP K homology) domains, consisting of ∼70 amino acid residues, are present in a variety of nucleic-acid-binding proteins. Among these are poly(C)-binding proteins (PCBPs), which are important regulators of mRNA stability and posttranscriptional regulation in general. All PCBPs contain three different KH domains and recognize poly(C)-sequences with high affinity and specificity. To reveal the molecular basis of poly(C)-sequence recognition, we have determined the crystal structure, at 1.6 Å resolution, of PCBP2 KH3 domain in complex with a 7-nt DNA sequence (5′-AACCCTA-3′) corresponding to one repeat of the C-rich strand of human telomeric DNA. The domain assumes a type-I KH fold in a βααββα configuration. The protein–DNA interface could be studied in unprecedented detail and is made up of a series of direct and water-mediated hydrogen bonds between the protein and the DNA, revealing an especially dense network involving several structural water molecules for the last 2 nt in the core recognition sequence. Unlike published KH domain structures, the protein crystallizes without protein–protein contacts, yielding new insights into the dimerization properties of different KH domains. A nucleotide platform, an interesting feature found in some RNA molecules, was identified, evidently for the first time in DNA.