Structure of a Construct of a Human Poly(C)-binding Protein Containing the First and Second KH Domains Reveals Insights into Its Regulatory Mechanisms

Abstract

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...

Four KH domains of the C. elegans Bicaudal-C ortholog GLD-3 form a globular structural platform

“…It is also similar to that observed in the crystals of isolated KH domains, for example, between symmetry-related Nova-1 KH3 domains (Lewis et al 1999) or symmetry-related PCBP2 KH1 domains (Supplemental Fig. 2C, right panel; Du et al 2005Du et al , 2008. The KH2-KH5 architecture of GLD-3 does not show the other type of interface reported from crystals of isolated KH domains, namely, a helix-helix packing between symmetry-related a3-helices as seen in Nova-2 KH3 (Lewis et al 1999;Valverde et al 2008).…”

Expression and functional role of a transcribed noncoding RNA with an ultraconserved element in hepatocellular carcinoma

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