桑迫 香奈子

Summary The pre-spliceosomal complex involves interactions between U1 and U2 snRNPs, where a ubiquitin-like domain (ULD) of SF3A1, a component of U2 snRNP, binds to the stem-loop 4 (SL4; the UUCG tetraloop) of U1 snRNA in U1 snRNP. Here, we reported the 1.80 Å crystal structure of human SF3A1 ULD (ULDSF3A1) complexed with SL4. The structural part of ULDSF3A1 (res. 704–785) adopts a typical β-grasp fold with a topology of β1-β2-α1-310a-β3-β4-310b-β5, closely resembling that of ubiquitin, except for the length and structure of the β1/β2 loop. A patch on the surface formed by three ULDSF3A1-specific residues, Lys756 (β3), Phe763 (β4), and Lys765 (following β4), contacts the canonical UUCG tetraloop structure. In contrast, the directly following C-terminal tail composed of 786KERGGRKK793 was essentially stretched. The main or side chains of all the residues interacted with the major groove of the stem helix; the RGG residues adopted a peculiar conformation for RNA recognition. These findings were confirmed by mutational studies using bio-layer interferometry. Collectively, a unique combination of the β-grasp fold and the C-terminal tail constituting ULDSF3A1 is required for the SL4-specific binding. This interaction mode also suggests that putative post-translational modifications, including ubiquitination in ULDSF3A1, directly inhibit SL4 binding.

<title>Abstract</title> The accurate exclusion of introns by RNA splicing is critical for the production of mature mRNA. U2AF1 binds specifically to the 3´ splice site, which includes an essential AG dinucleotide. Even a single amino acid mutation of U2AF1 can cause serious disease such as certain cancers or myelodysplastic syndromes. Here, we describe the first crystal structures of wild-type and pathogenic mutant U2AF1 complexed with target RNA, revealing the mechanism of 3´ splice site selection, and how aberrant splicing results from clinically important mutations. Unexpected features of this mechanism may assist the future development of new treatments against diseases caused by splicing errors.

The spliceosomal protein SF3b49, a component of the splicing factor 3b (SF3b) protein complex in the U2 small nuclear ribonucleoprotein, contains two RNA recognition motif (RRM) domains. In yeast, the first RRM domain (RRM1) of Hsh49 protein (yeast orthologue of human SF3b49) reportedly interacts with another component, Cus1 protein (orthologue of human SF3b145). Here, we solved the solution structure of the RRM1 of human SF3b49 and examined its mode of interaction with a fragment of human SF3b145 using NMR methods. Chemical shift mapping showed that the SF3b145 fragment spanning residues 598-631 interacts with SF3b49 RRM1, which adopts a canonical RRM fold with a topology of 1-1-2-3-2-4. Furthermore, a docking model based on NOESY measurements suggests that residues 607-616 of the SF3b145 fragment adopt a helical structure that binds to RRM1 predominantly via 1, consequently exhibiting a helix-helix interaction in almost antiparallel. This mode of interaction was confirmed by a mutational analysis using GST pull-down assays. Comparison with structures of all RRM domains when complexed with a peptide found that this helix-helix interaction is unique to SF3b49 RRM1. Additionally, all amino acid residues involved in the interaction are well conserved among eukaryotes, suggesting evolutionary conservation of this interaction mode between SF3b49 RRM1 and SF3b145. PDB Code(s):

The pre-mRNA splicing reaction of eukaryotic cells has to be carried out extremely accurately, as failure to recognize the splice sites correctly causes serious disease. The small subunit of the U2AF heterodimer is essential for the determination of 3 ' splice sites in pre-mRNA splicing, and several single-residue mutations of the U2AF small subunit cause severe disorders such as myelodysplastic syndromes. However, the mechanism of RNA recognition is poorly understood. Here we solved the crystal structure of the U2AF small subunit (U2AF23) from fission yeast, consisting of an RNA recognition motif (RRM) domain flanked by two conserved CCCH-type zinc fingers (ZFs). The two ZFs are positioned side by side on the beta sheet of the RRM domain. Further mutational analysis revealed that the ZFs bind cooperatively to the target RNA sequence, but the RRM domain acts simply as a scaffold to organize the ZFs and does not itself contact the RNA directly. This completely novel and unexpected mode of RNA-binding mechanism by the U2AF small subunit sheds light on splicing errors caused by mutations of this highly conserved protein.

D-Serine is an essential coagonist with glutamate for stimulation of N-methyl-D-aspartate (NMDA) glutamate receptors. Although astrocytic metabolic processes are known to regulate synaptic glutamate levels, mechanisms that control D-serine levels are not well defined. Here we show that D-serine production in astrocytes is modulated by the interaction between the D-serine synthetic enzyme serine racemase (SRR) and a glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In primary cultured astrocytes, glycolysis activity was negatively correlated with D-serine level. We show that SRR interacts directly with GAPDH, and that activation of glycolysis augments this interaction. Biochemical assays using mutant forms of GAPDH with either reduced activity or reduced affinity to SRR revealed that GAPDH suppresses SRR activity by direct binding to GAPDH and through NADH, a product of GAPDH. NADH allosterically inhibits the activity of SRR by promoting the disassociation of ATP from SRR. Thus, astrocytic production of D-serine is modulated by glycolytic activity via interactions between GAPDH and SRR. We found that SRR is expressed in astrocytes in the subiculum of the human hippocampus, where neurons are known to be particularly vulnerable to loss of energy. Collectively, our findings suggest that astrocytic energy metabolism controls D-serine production, thereby influencing glutamatergic neurotransmission in the hippocampus.

We developed fluorescent turn-on probes containing a fluorescent nucleoside, 5-(benzofuran-2-yl) deoxyuridine (dU(BF)) or 5-(3-methylbenzofuran-2-yl) deoxyuridine (dU(MBF)), for the detection of single-stranded DNA or RNA by utilizing DNA triplex formation. Fluorescence measurements revealed that the probe containing dUMBF achieved superior fluorescence enhancement than that containing dU(BF). NMR and fluorescence analyses indicated that the fluorescence intensity increased upon triplex formation partly as a consequence of a conformational change at the bond between the 3-methylbenzofuran and uracil rings. In addition, it is suggested that the microenvironment around the 3-methylbenzofuran ring contributed to the fluorescence enhancement. Further, we developed a method for detecting RNA by rolling circular amplification in combination with triplex-induced fluorescence enhancement of the oligonucleotide probe containing dU(MBF).

The family of cytoplasmic polyadenylation element binding proteins CPEB1, CPEB2, CPEB3, and CPEB4 binds to the 3'-untranslated region (3'-UTR) of mRNA, and plays significant roles in mRNA metabolism and translation regulation. They have a common domain organization, involving two consecutive RNA recognition motif (RRM) domains followed by a zinc finger domain in the C-terminal region. We solved the solution structure of the first RRM domain (RRM1) of human CPEB3, which revealed that CPEB3 RRM1 exhibits structural features distinct from those of the canonical RRM domain. Our structural data provide important information about the RNA binding ability of CPEB3 RRM1. (C) 2014 Wiley Periodicals, Inc.

Tissue-specific alternative pre-mRNA splicing is often cooperatively regulated by multiple splicing factors, but the structural basis of cooperative RNA recognition is poorly understood. In Caenorhabditis elegans, ligand binding specificity of fibroblast growth factor receptors (FGFRs) is determined by mutually exclusive alternative splicing of the sole FGFR gene, egl-15. Here we determined the solution structure of a ternary complex of the RNA-recognition motif (RRM) domains from the RBFOX protein ASD-1, SUP-12 and their target RNA from egl-15. The two RRM domains cooperatively interact with the RNA by sandwiching a G base to form the stable complex. Multichromatic fluorescence splicing reporters confirmed the requirement of the G and the juxtaposition of the respective cis elements for effective splicing regulation in vivo. Moreover, we identified a new target for the heterologous complex through an element search, confirming the functional significance of the intermolecular coordination.

The WWE domain is often identified in proteins associated with ubiquitination or poly-ADP-ribosylation. Structural information about WWE domains has been obtained for the ubiquitination-related proteins, such as Deltex and RNF146, but not yet for the poly-ADP-ribose polymerases (PARPs). Here we determined the solution structures of the WWE domains from PARP11 and PARP14, and compared them with that of the RNF146 WWE domain. NMR perturbation experiments revealed the specific differences in their ADP-ribose recognition modes that correlated with their individual biological activities. The present structural information sheds light on the ADP-ribose recognition modes by the PARP WWE domains. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Human Transformer2-beta (hTra2-beta) is an important member of the serine/arginine-rich protein family, and contains one RNA recognition motif (RRM). It controls the alternative splicing of several pre-mRNAs, including those of the calcitonin/calcitonin gene-related peptide (CGRP), the survival motor neuron 1 (SMN1) protein and the tau protein. Accordingly, the RRM of hTra2-beta specifically binds to two types of RNA sequences [the CAA and (GAA)(2) sequences]. We determined the solution structure of the hTra2-beta RRM (spanning residues Asn110-Thr201), which not only has a canonical RRM fold, but also an unusual alignment of the aromatic amino acids on the beta-sheet surface. We then solved the complex structure of the hTra2-beta RRM with the (GAA)(2) sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the beta-sheet surface. Further NMR experiments revealed that the hTra2-beta RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-beta RRM recognizes two types of RNA sequences in different RNA binding modes.

The zinc finger CW (zf-CW) domain is a motif of about 60 residues that is frequently found in proteins involved in epigenetic regulation. Here, we determined the NMR solution structure of the zf-CW domain of the human zf-CW and PWWP domain containing protein 1 (ZCWPW1). The zf-CW domain adopts a new fold in which a zinc ion is coordinated tetrahedrally by four conserved Cys ligand residues. The tertiary structure of the zf-CW domain partially resembles that adopted by the plant homeo domain (PHD) finger bound to the histone tail, suggesting that the zf-CW domain and the PHD finger have similar functions. The solution structure of the complex of the zf-CW domain with the histone H3 tail peptide (1-10) with trimethylated K4 clarified its binding mode. Our structural and biochemical studies have identified the zf-CW domain as a member of the histone modification reader modules for epigenetic regulation.

The CUG-binding protein 1 (CUG-BP1) is a member of the CUG-BP1 and ETR-like factors (CELF) family or the Bruno-like family and is involved in the control of splicing, translation and mRNA degradation. Several target RNA sequences of CUG-BP1 have been predicted, such as the CUG triplet repeat, the GU-rich sequences and the AU-rich element of nuclear pre-mRNAs and/or cytoplasmic mRNA. CUG-BP1 has three RNA-recognition motifs (RRMs), among which the third RRM (RRM3) can bind to the target RNAs on its own. In this study, we solved the solution structure of the CUG-BP1 RRM3 by hetero-nuclear NMR spectroscopy. The CUG-BP1 RRM3 exhibited a noncanonical RRM fold, with the four-stranded beta-sheet surface tightly associated with the N-terminal extension. Furthermore, we determined the solution structure of the CUG-BP1 RRM3 in the complex with (UG)(3) RNA, and discovered that the UGU trinucleotide is specifically recognized through extensive stacking interactions and hydrogen bonds within the pocket formed by the beta-sheet surface and the N-terminal extension. This study revealed the unique mechanism that enables the CUG-BP1 RRM3 to discriminate the short RNA segment from other sequences, thus providing the molecular basis for the comprehension of the role of the RRM3s in the CELF/Bruno-like family.

The human AU RNA binding protein/enoyl-Coenzyme A hydratase (AUH) is a 3-hydroxy-3-methylglutaconyl-CoA dehydratase in the leucine degradation pathway. It also possesses an RNA-binding activity to AUUU repeats, which involves no known conserved RNA-binding domains and is seemingly unrelated to the enzymatic activity. In this study, we performed mass spectrometric analyses to elucidate the oligomeric states of AUH in the presence and absence of RNA. With a short RNA (AUUU) or without RNA, AUH mainly exists as a trimer in solution. On the other hand, the AUH trimer dimerizes upon binding to one molecule of a long RNA containing 24 repeats of the AUUU motif, (AUUU)(24)A. AUH was crystallized with the long RNA. Although the RNA was disordered in the crystalline lattice, the AUH structure was determined as an asymmetric dimer of trimers with a kink in the alignment of the trimer axes, resulting in the formation of two clefts with significantly different sizes.

T cell intracellular antigen-1 (TIA-1), an apoptosis promoting factor, functions as a splicing regulator for the Fas pre-mRNA. TIA-1 possesses three RNA recognition motifs (RRMs) and a glutamine-rich domain. The second RRM (RRM2) is necessary and sufficient for tight, sequence-specific binding to the uridine-rich sequences buried around the 5' splice sites. In the present study, we solved the solution structure of the murine TIA-1 RRM2 by heteronuclear-nuclear magnetic resonance spectroscopy. The TIA-1 RRM2 adopts the RRM fold (beta alpha beta beta alpha beta) and possesses an extra beta-strand between beta 2 and beta 3, which forms an additional beta-sheet with the C-terminal part of beta 2. We refer to this structure as the beta 2-beta 2' beta-loop. Interestingly, this characteristic beta-loop structure is conserved among a number of RRMs, including the U2AF65 RRM2 and the Sex-lethal RRM1 and RRM2, which also bind to uridine-rich RNAs. Furthermore, we identified a new sequence motif in the beta 2-beta 2' beta-loop, the DxxT motif. Chemical shift perturbation analyses of both the main and side chains upon binding to the uridine pentamer RNA revealed that most of the beta-sheet surface, including the beta 2-beta 2' beta-loop, is involved in the RNA binding. An investigation of the chemical shift perturbation revealed similarity in the RNA recognition modes between the TIA-1 and U2AF65 RRMs.

The spliceosomal protein p14, a component of the SF3b complex in the U2 small nuclear ribonucleoprotein.(snRNP), is essential for the U2 snRNP to recognize the branch site adenosine. The elucidation of the dynamic process of the splicing machinery rearrangement awaited the solution structural information. We identified a suitable complex of human p14 and the SF3b155 fragment for the determination of its solution structure by NMR. In addition to the overall structure Of the complex, which was recently reported in a crystallographic study (typical RNA recognition motif fold beta 1-alpha 1-beta 2-beta 3-alpha 2-beta 4 of p14, and alpha A-beta A fold of the SF3b155 fragment), we identified three important features revealed by the NMR solution structure. First, the C-terminal extension and. the nuclear localization signal of p14 (alpha 3 and alpha 4 in the crystal structure, respectively) were dispensable for the complex formation. Second, the proline-rich segment of SF3b155, following beta A, closely approaches p14. Third, interestingly, the beta 1-alpha 1 loop and the alpha 2-beta 4 beta-hairpin form a positively charged groove. Extensive mutagenesis analyses revealed the functional relevance of the residues involved in the protein-protein interactions: two aromatic residues of SF3b155 (Phe408 and Tyr412) play crucial roles in the complex formation, and two hydrophobic residues (Val414 and Leu415) in SF3b 155 serve as an anchor for the complex formation, by cooperating with the aromatic residues. These findings clearly led to the conclusion that SFb155 binds to p14 with three contact points, involving Phe408, Tyr412, and Val414/Leu415. Furthermore, to dissect the interactions between p 14 and the branch site RNA, we performed chemical-shift-perturbation experiments, not only for the main-chain but also for the side-chain resonances, for several p14-SF3b155 complex constructs upon binding to RNA. These analyses identified a positively charged groove and the C-terminal extension of p14 as RNA-binding sites. Strikingly, an aromatic residue in the beta 1-alpha 1 loop, Tyr28, and a positively charged residue in the alpha 2-beta 4 beta-hairpin, Agr85, are critical for the RNA-binding activity of the positively charged groove. The 7yr28Ala and Arg85Ala point mutants and a deletion mutant of the C-terminal extension clearly revealed that their RNA binding activities were independent of each other. Collectively, this study provides details for the protein-recognition mode of p14 and insight into the branch site recognition.

The SF3a complex, consisting of SF3a60, SF3a66, and SF3a120, in 17S U2 snRNP is crucial to spliceosomal assembly. SF3a120 contains two tandem SURP domains (SURP1 and SURP2), and SURP2 is responsible for binding to SF3a60. We found that the SURP2 fragment forms a stable complex with an SF3a60 fragment (residues 71-107) and solved its structure by NMR spectroscopy. SURP2 exhibits a fold of the alpha 1-alpha 2-alpha 3(10)-alpha 3 topology, and the SF3a60 fragment forms an amphipathic alpha helix intimately contacting all of SURP2. We also solved the SURP1 structure, which has the same fold as SURP2. The protein-binding interface of SURP2 is quite similar to the corresponding surface of SURP1, except for two amino acid residues. One of them, Leu169, is characteristic of SF3a120 SURP2 among SURP domains. Mutagenesis showed that this single Leu residue is the critical determinant for complex formation, which reveals the protein recognition mechanism in the subunit assembly.

Previously, we established for the first time an in vivo monitoring assay system conjugated with random mutagenesis in order to study the structure-function relationship of the antimicrobial peptide, apidaecin [Taguchi ct al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, this methodology was used to carry out the functional mapping of a second target, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum ct al. (1996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene encoding thanatin was expressed in a fused form with Streptomyces protease inhibitor protein, SSI, under the control of tac promoter in Escherichia coli JM109. Expression of the thanatin-fused protein was found to depend on the concentration of the transcriptional inducer, isopropyl-beta -D-thio-galactopyranoside (IPTG), and to parallel the degree of growth inhibition of the transformant cells. When a PCR random mutation was introduced into the structural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colony size (plate assay) or optical density (liquid assay) in comparison with the wild-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by the in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubly mutated protein (K17R/R20G), were stably overproduced with a fusion. partner protein resulting in the efficient formation of inclusion bodies in E. coli BL21(DE3). The products were isolated in large amounts (yield 30%) from the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coli JM109 activities, judged by minimum inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on the NMR solution structure and molecular dynamics, the structure- function relationship of thanatin is discussed by comparing the functional mapping data obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining the beta -strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity.