The modulation of pre‐mRNA splicing is proposed as an attractive anti‐neoplastic strategy, especially for the cancers that exhibit aberrant pre‐mRNA splicing. Here, we discovered that T‐025 functions as an orally available and potent inhibitor of Cdc2‐like kinases (CLKs), evolutionally conserved kinases that facilitate exon recognition in the splicing machinery. Treatment with T‐025 reduced CLK‐dependent phosphorylation, resulting in the induction of skipped exons, cell death, and growth suppression in vitro and in vivo. Further, through growth inhibitory characterization, we identified high CLK2 expression or MYC amplification as a sensitive‐associated biomarker of T‐025. Mechanistically, the level of CLK2 expression correlated with the magnitude of global skipped exons in response to T‐025 treatment. MYC activation, which altered pre‐mRNA splicing without the transcriptional regulation of CLKs, rendered cancer cells vulnerable to CLK inhibitors with synergistic cell death. Finally, we demonstrated in vivo anti‐tumor efficacy of T‐025 in an allograft model of spontaneous, MYC‐driven breast cancer, at well‐tolerated dosage. Collectively, our results suggest that the novel CLK inhibitor could have therapeutic benefits, especially for MYC‐driven cancer patients.

Recognition of poly(C) DNA and RNA sequences in mammalian cells is achieved by a subfamily of the KH (hnRNP K homology) domain-containing proteins known as poly(C)-binding proteins (PCBPs).To reveal the molecular basis of poly(C) sequence recognition, we have determined the crystal structure, at 1.7-Å resolution, of PCBP2 KH1 in complex with a 7-nucleotide DNA sequence (5-AACCCTA-3) corresponding to one repeat of the human C-rich strand telomeric DNA. The protein-DNA interaction is mediated by the combination of several stabilizing forces including hydrogen bonding, electrostatic interactions, van der Waals contacts, and shape complementarities. Specific recognition of the three cytosine residues is realized by a dense network of hydrogen bonds involving the side chains of two conserved lysines and one glutamic acid. The co-crystal structure also reveals a protein-protein dimerization interface of PCBP2 KH1 located on the opposite side of the protein from the DNA binding groove. Numerous stabilizing protein-protein interactions, including hydrophobic contacts, stacking of aromatic side chains, and a large number of hydrogen bonds, indicate that the protein-protein interaction interface is most likely genuine. Interaction of PCBP2 KH1 with the C-rich strand of human telomeric DNA suggests that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.K-homology domain (KH 2 domain, originally identified in hnRNP-K) is one of the most frequently occurring and conserved nucleic acidbinding protein motifs. So far, a large number of KH domain-containing proteins have been identified in a wide variety of species ranging from bacteria to human. These KH domain proteins assume a wide spectrum of biological functions, including transcriptional and translational controls, mRNA stabilization, and mRNA splicing, among others. Different KH domains possess quite different nucleic acid binding specificities. Unveiling how different KH domains interact specifically with their nucleic acid targets and how these interactions contribute to the complex regulatory processes is of central importance to a better understanding of how KH domain proteins function.One of the most distinctive nucleic acid binding specificities achieved by the KH domains is manifested by a subfamily of KH domain-containing proteins known as poly(C)-binding proteins (PCBPs). As implied by the family name, PCBPs recognize poly(C) RNA or DNA sequences with high affinity and specificity (for reviews, see Refs. 1 and 2). To date, five evolutionarily related PCBPs have been identified in mammalian cells: PCBP1-4 (also known as ␣CP1-4; PCBP1 and -2 are also known as hnRNP E1 and E2) and hnRNP K. Each PCBP contains three KH domains: two consecutive KH domains at the N terminus and a third KH domain at the C terminus with an intervening sequence of variable length between the second and third KH domains (Fig. 1A). In general, corresponding KH domains share a higher degree of homology than KH domains within each protein (Fig. 1B)...

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...

Dicer, an RNase III enzyme, initiates RNA interference by processing precursor dsRNAs into mature microRNAs and small-interfering RNAs. It is also involved in loading and activation of the RNAinduced silencing complex. Here, we report the crystal structures of a catalytically active fragment of mouse Dicer, containing the RNase IIIb and dsRNA binding domains, in its apo and Cd 2؉ -bound forms, at 1.68-and 2.8-Å resolution, respectively. Models of this structure with dsRNA reveal that a lysine residue, highly conserved in Dicer RNase IIIa and IIIb domains and in Drosha RNase IIIb domains, has the potential to participate in the phosphodiester bond cleavage reaction by stabilizing the transition state and leaving group of the scissile bond. Mutational and enzymatic assays confirm the importance of this lysine in dsRNA cleavage, suggesting that this lysine represents a conserved catalytic residue of Dicers. The structures also reveals a Ϸ45-aa region within the RNase IIIb domain that harbors an ␣-helix at the N-terminal half and a flexible loop at the C-terminal half, features not present in previously reported structures of homologous RNase III domains from either bacterial RNase III enzymes or Giardia Dicer. N-terminal residues of this ␣-helix have the potential to engage in minor groove interaction with dsRNA substrates.dsRNA processing ͉ RNA interference ͉ RNase III ͉ x-ray crystallography T he innate gene silencing mechanism of RNA interference (RNAi) is triggered by small dsRNAs (1, 2). Fundamental roles of RNAi include defense against viruses (3), regulation of development (4), and genome maintenance (5, 6). The two major classes of small RNAs responsible for this mechanism are microRNAs (miRNAs) and small-interfering RNAs (siRNAs) (7, 8); both are generated by the riboendonuclease Dicer (9, 10).Dicer processes precursor miRNAs (pre-miRNA, generated by the riboendonuclease Drosha in the nucleus from the primary transcripts of miRNAs) and long dsRNAs (generated during viral infections or introduced experimentally into the cell) into mature miRNAs/siRNAs. It then loads the miRNA/siRNA into the Argonaute-containing RNAi effector complex RNAinduced silencing complex (RISC) (11). The passenger strand of the miRNA/siRNA is cleaved by Argonaute and discarded (12, 13); the guide strand leads RISC to target mRNAs with sequence motifs complementary to that of the guide. The result is either mRNA degradation or translational suppression (10, 14). Structures of Argonaute and related proteins have contributed to these insights into the mechanisms of Argonaute activities (for reviews see refs. 15 and 16 and references therein).Dicer is an RNase III enzyme specific for dsRNAs. RNase III cleavage products contain 5Ј phosphate and 3Ј hydroxyl termini and a 2-nt overhang at the 3Ј end. Dicer products are also characterized by its discrete size of Ϸ21 nt (17). RNase III enzymes can be divided into three classes (Fig. 1). Class I enzymes, found in bacteria, bacteriophage, and fungi, contain a single RNase III domain and a dsRNA...