Epitopes recognition of SARS-CoV-2 nucleocapsid RNA binding domain by human monoclonal antibodies
Youngchang KimN. MaltsevaC. TesarR. JedrzejczakM. EndresHeng MaHaley L. DuganChristopher T. StamperChangsoo ChangLei LiSiriruk ChangrobNai‐Ying ZhengMin HuangArvind RamanathanPatrick C. WilsonK. MichalskaA. Joachimiak
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Coronavirus nucleocapsid protein (NP) of SARS-CoV-2 plays a central role in many functions important for virus proliferation including packaging and protecting genomic RNA. The protein shares sequence, structure, and architecture with nucleocapsid proteins from betacoronaviruses. The N-terminal domain (NPKeywords:
Coronavirus
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C2 domain
Factor V
Prothrombinase
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Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca2+-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca2+. Exon 2 encodes an α-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.
Amino terminal
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Two actin‐binding sites have been identified on human dystrophin by proton NMR spectroscopy of synthetic peptides corresponding to defined regions of the polypeptide sequence. These are Actin‐Binding Site 1 (ABS1) located at residues 17–26 and Actin‐Binding Site 2 (ABS2) in the region of residues 128–156. Using defined fragments of the actin amino acid sequence, ABS1 has been shown to bind to actin in the region represented by residues 83–117 and ABS2 to the C‐terminal region represented by residues 350–375. These dystrophin‐binding sites lie on the exposed domain in the actin filament.
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Actin-binding protein
Actina
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Mmi1 is an essential RNA-binding protein in the fission yeast Schizosaccharomyces pombe that eliminates meiotic transcripts during normal vegetative growth. Mmi1 contains a YTH domain that binds specific RNA sequences, targeting mRNAs for degradation. The YTH domain of Mmi1 uses a noncanonical RNA-binding surface that includes contacts outside the conserved fold. Here, we report that an N-terminal extension that is proximal to the YTH domain enhances RNA binding. Using X-ray crystallography, NMR, and biophysical methods, we show that this low-complexity region becomes more ordered upon RNA binding. This enhances the affinity of the interaction of the Mmi1 YTH domain with specific RNAs by reducing the dissociation rate of the Mmi1–RNA complex. We propose that the low-complexity region influences RNA binding indirectly by reducing dynamic motions of the RNA-binding groove and stabilizing a conformation of the YTH domain that binds to RNA with high affinity. Taken together, our work reveals how a low-complexity region proximal to a conserved folded domain can adopt an ordered structure to aid nucleic acid binding. Mmi1 is an essential RNA-binding protein in the fission yeast Schizosaccharomyces pombe that eliminates meiotic transcripts during normal vegetative growth. Mmi1 contains a YTH domain that binds specific RNA sequences, targeting mRNAs for degradation. The YTH domain of Mmi1 uses a noncanonical RNA-binding surface that includes contacts outside the conserved fold. Here, we report that an N-terminal extension that is proximal to the YTH domain enhances RNA binding. Using X-ray crystallography, NMR, and biophysical methods, we show that this low-complexity region becomes more ordered upon RNA binding. This enhances the affinity of the interaction of the Mmi1 YTH domain with specific RNAs by reducing the dissociation rate of the Mmi1–RNA complex. We propose that the low-complexity region influences RNA binding indirectly by reducing dynamic motions of the RNA-binding groove and stabilizing a conformation of the YTH domain that binds to RNA with high affinity. Taken together, our work reveals how a low-complexity region proximal to a conserved folded domain can adopt an ordered structure to aid nucleic acid binding.
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Regulation of the CFTR Cl channel function involves a protein complex of activated protein kinase Cε (PKCε) bound to RACK1, a receptor for activated C kinase, and RACK1 bound to the human Na+/H+ exchanger regulatory factor (NHERF1) in human airway epithelial cells. Binding of NHERF1 to RACK1 is mediated via a NHERF1−PDZ1 domain. The goal of this study was to identify the binding motif for human NHERF1 on RACK1. We examined the site of binding of NHERF1 on RACK1 using peptides encoding the seven WD40 repeat units of human RACK1. One WD repeat peptide, WD5, directly binds NHERF1 and the PDZ1 domain with similar EC50 values, blocks binding of recombinant RACK1 and NHERF1, and pulls down endogenous RACK1 from Calu-3 cell lysate in a dose-dependent manner. The remaining WD repeat peptides did not block RACK1−NHERF1 binding. An 11-amino acid peptide encoding a site on the PDZ1 domain blocks binding of the WD5 repeat peptide with the PDZ1 domain. An N-terminal 12-amino acid segment of the WD5 repeat peptide, which comprises the first of four antiparallel β-strands, dose-dependently binds to the PDZ1 domain of NHERF1 and blocks binding of the PDZ1 domain to RACK1. These results suggest that the binding site might form a β-turn with topology sufficient for binding of NHERF1. Our results also demonstrate binding of NHERF to RACK1 at the WD5 repeat, which is distinct from the PKCε binding site on the WD6 repeat of RACK1.
A-site
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The RNA binding proteins (RBPs) human antigen R (HuR) and Tristetraprolin (TTP) are known to exhibit competitive binding but have opposing effects on the bound messenger RNA (mRNA). How cells discriminate between the two proteins is an interesting problem. Machine learning approaches, such as support vector machines (SVMs), may be useful in the identification of discriminative features. However, this method has yet to be applied to studies of RNA binding protein motifs.Applying the k-spectrum kernel to a support vector machine (SVM), we first verified the published binding sites of both HuR and TTP. Additional feature engineering highlighted the U-rich binding preference of HuR and AU-rich binding preference for TTP. Domain adaptation along with multi-task learning was used to predict the common binding sites.The distinction between HuR and TTP binding appears to be subtle content features. HuR prefers strongly U-rich sequences whereas TTP prefers AU-rich as with increasing A content, the sequences are more likely to be bound only by TTP. Our model is consistent with competitive binding of the two proteins, particularly at intermediate AU-balanced sequences. This suggests that fine changes in the A/U balance within a untranslated region (UTR) can alter the binding and subsequent stability of the message. Both feature engineering and domain adaptation emphasized the extent to which these proteins recognize similar general sequence features. This work suggests that the k-spectrum kernel method could be useful when studying RNA binding proteins and domain adaptation techniques such as feature augmentation could be employed particularly when examining RBPs with similar binding preferences.
Tristetraprolin
Discriminative model
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Previous evidence suggests multiple anesthetic binding sites on human serum albumin, but to date, we have only identified Trp-214 in an interdomain cleft as contributing to a binding site. We used a combination of site-directed mutagenesis, photoaffinity labeling, amide hydrogen exchange, and tryptophan fluorescence spectroscopy to evaluate the importance to binding of a large domain III cavity and compare it to binding character of the 214 interdomain cleft. The data show anesthetic binding in this domain III cavity of similar character to the interdomain cleft, but selectivity for different classes of anesthetics exists. Occupancy of these sites stabilizes the native conformation of human serum albumin. The features necessary for binding in the cleft appear to be fairly degenerate, but in addition to hydrophobicity, there is evidence for the importance of polarity. Finally, myristate isosterically competes with anesthetic binding in the domain III cavity and allosterically enhances anesthetic binding in the interdomain cleft.
Human serum albumin
Amide
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Sam68 is an RNA-binding protein that contains a heterogeneous nuclear ribonucleoprotein K homology domain embedded in a larger RNA binding domain called the GSG (GRP33,Sam68, GLD-1) domain. This family of proteins is often referred to as the STAR (signaltransduction and activators of RNA metabolism) proteins. It is not known whether Sam68 is a general nonspecific RNA-binding protein or whether it recognizes specific response elements in mRNAs with high affinity. Sam68 has been shown to bind homopolymeric RNA and a synthetic RNA sequence called G8–5 that has a core UAAA motif. Here we performed a structure function analysis of Sam68 and identified two arginine glycine (RG)-rich regions that confer nonspecific RNA binding to the Sam68 GSG domain. In addition, by using chimeric proteins between Sam68 and QKI-7, we demonstrated that one of the Sam68 RG-rich sequences of 26 amino acids was sufficient to confer homopolymeric RNA binding to the GSG domain of QKI-7, another STAR protein. Furthermore, that minimal sequence can also give QKI-7 the ability (as Sam68) to functionally substitute for HIV-1 REV to facilitate the nuclear export of RNAs. Our studies suggest that neighboring RG-rich sequences may impose nonspecific RNA binding to GSG domains. Because the Sam68 RNA binding activity is negatively regulated by tyrosine phosphorylation, our data lead us to propose that Sam68 might be a specific RNA-binding protein when tyrosine phosphorylated. Sam68 is an RNA-binding protein that contains a heterogeneous nuclear ribonucleoprotein K homology domain embedded in a larger RNA binding domain called the GSG (GRP33,Sam68, GLD-1) domain. This family of proteins is often referred to as the STAR (signaltransduction and activators of RNA metabolism) proteins. It is not known whether Sam68 is a general nonspecific RNA-binding protein or whether it recognizes specific response elements in mRNAs with high affinity. Sam68 has been shown to bind homopolymeric RNA and a synthetic RNA sequence called G8–5 that has a core UAAA motif. Here we performed a structure function analysis of Sam68 and identified two arginine glycine (RG)-rich regions that confer nonspecific RNA binding to the Sam68 GSG domain. In addition, by using chimeric proteins between Sam68 and QKI-7, we demonstrated that one of the Sam68 RG-rich sequences of 26 amino acids was sufficient to confer homopolymeric RNA binding to the GSG domain of QKI-7, another STAR protein. Furthermore, that minimal sequence can also give QKI-7 the ability (as Sam68) to functionally substitute for HIV-1 REV to facilitate the nuclear export of RNAs. Our studies suggest that neighboring RG-rich sequences may impose nonspecific RNA binding to GSG domains. Because the Sam68 RNA binding activity is negatively regulated by tyrosine phosphorylation, our data lead us to propose that Sam68 might be a specific RNA-binding protein when tyrosine phosphorylated. Src associated substrate in mitosis of68 kDa heterogeneous nuclear ribonucleoproteinK homology germlinedefective-1 glycine-rich protein of 33kDa GRP33, Sam68, GLD-1 N-terminal of KH domain C-terminal of KH domain signaltransduction and activator of RNA metabolism quaking polymerase chain reaction untranslated region hemagglutinin polyacrylamide gel electrophoresis REV response element chloramphenicol acetyltransferase Sam68 (Src substrate associated duringmitosis of 68 kDa)1 is a substrate for tyrosine kinases including Src family kinases p60src (1Wong G. Muller O. Clark R. Conroy L. Moran M.F. Polakis P. McCormick F. Cell. 1992; 69: 551-558Abstract Full Text PDF PubMed Scopus (226) Google Scholar, 2Taylor S.J. Shalloway D. Nature. 1994; 368: 867-871Crossref PubMed Scopus (373) Google Scholar, 3Fumagalli S. Totty N.F. Hsuan J.J. Courtneidge S.A. Nature. 1994; 368: 871-874Crossref PubMed Scopus (324) Google Scholar, 4Weng Z. Thomas S.M. Rickles R.J. Taylor J.A. Brauer A.W. Seidel-Dugan C. Michael W.M. Dreyfuss G. Brugge J.S. Mol. Cell. Biol. 1994; 14: 4509-4521Crossref PubMed Scopus (206) Google Scholar), p59fyn (5Richard S., Yu, D. Blumer K.J. Hausladen D. Olszowy M.W. Connelly P.A. Shaw A.S. Mol. Cell. Biol. 1995; 15: 186-197Crossref PubMed Google Scholar), p56lck (6Vogel L.B. Fujita D.J. J. Biol. Chem. 1995; 270: 2506-2511Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), BRK/SIK (7Derry J.J. Richard S. Valderrama Carvajal H. Ye X. Vasioukhin V. Cochrane A.W. Chen T. Tyner A.L. Mol. Cell. Biol. 2000; 20: 6114-6126Crossref PubMed Scopus (134) Google Scholar), and ZAP70 (8Lang V. Mege D. Semichon M. Gary-Gouy H. Bismuth G. Eur. J. Immunol. 1997; 27: 3360-3367Crossref PubMed Scopus (23) Google Scholar). Sam68 has been shown to bind numerous Src homology 3, Src homology 2, and WW domain-containing proteins, leading several groups to suggest that Sam68 may be an adaptor protein for tyrosine kinases (5Richard S., Yu, D. Blumer K.J. Hausladen D. Olszowy M.W. Connelly P.A. Shaw A.S. Mol. Cell. Biol. 1995; 15: 186-197Crossref PubMed Google Scholar, 9Taylor S.J. Anafi M. Pawson T. Shalloway D. J. Biol. Chem. 1995; 270: 10120-10124Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Sam68 is an RNA-binding protein that contains a KH domain embedded in a larger domain of ∼200 amino acids, the GSG (GRP33,Sam68, and GLD-1) domain. Sam68 has been shown to bind homopolymeric RNA poly(U) and poly(A) (2Taylor S.J. Shalloway D. Nature. 1994; 368: 867-871Crossref PubMed Scopus (373) Google Scholar, 10Chen T. Damaj B.B. Herrera C. Lasko P. Richard S. Mol. Cell. Biol. 1997; 17: 5707-5718Crossref PubMed Scopus (163) Google Scholar). The tyrosine phosphorylation of Sam68 by p59fyn severely inhibits its ability to bind poly(U)-Sepharose (11Wang L.L. Richard S. Shaw A.S. J. Biol. Chem. 1995; 270: 2010-2013Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Sam68 has also been shown to bind synthetic RNA sequences with a core UAAA with high affinity (12Lin Q. Taylor S.J. Shalloway D. J. Biol. Chem. 1997; 272: 27274-27280Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The function of Sam68 is unknown, but recent findings suggest that it may be involved in the regulation of splicing and/or RNA transport (13Chen T. Boisvert F.M. Bazett-Jones D.P. Richard S. Mol. Biol. Cell. 1999; 10: 3015-3033Crossref PubMed Scopus (124) Google Scholar, 14Reddy T.R. Xu W. Mau J.K. Goodwin C.D. Suhasini M. Tang H. Frimpong K. Rose D.W. Wong-Staal F. Nat. Med. 1999; 5: 635-642Crossref PubMed Scopus (146) Google Scholar, 15Bedford M.T. Frankel A. Yaffe M.B. Clarke S. Leder P. Richard S. J. Biol. Chem. 2000; 275: 16030-16036Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 16Hartmann A.M. Nayler O. Schwaiger F.W. Obermeier A. Stamm S. Mol. Biol. Cell. 1999; 10: 3909-3926Crossref PubMed Scopus (162) Google Scholar). The GSG domain is an evolutionarily conserved protein module initially identified by aligning the first three members of this family (17Jones A.R. Schedl T. Genes Dev. 1995; 9: 1491-1504Crossref PubMed Scopus (220) Google Scholar, 18Di Fruscio M. Chen T. Bonyadi S. Lasko P. Richard S. J. Biol. Chem. 1998; 273: 30122-30130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In addition to the KH domain, the GSG domain contains ∼75 amino acids N-terminal and ∼25 amino acids C-terminal to the KH domain called the NK (N-terminal of KH) and CK (C-terminal of KH) regions, respectively (schematically represented in Fig. 1). Several properties have been ascribed to the GSG domain including RNA binding (10Chen T. Damaj B.B. Herrera C. Lasko P. Richard S. Mol. Cell. Biol. 1997; 17: 5707-5718Crossref PubMed Scopus (163) Google Scholar, 12Lin Q. Taylor S.J. Shalloway D. J. Biol. Chem. 1997; 272: 27274-27280Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 18Di Fruscio M. Chen T. Bonyadi S. Lasko P. Richard S. J. Biol. Chem. 1998; 273: 30122-30130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 19Chen T. Richard S. Mol. Cell. Biol. 1998; 18: 4863-4871Crossref PubMed Scopus (105) Google Scholar, 20Di Fruscio M. Chen T. Richard S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2710-2715Crossref PubMed Scopus (91) Google Scholar, 21Rain J.C. Rafi Z. Rhani Z. Legrain P. Kramer A. RNA (N. Y.). 1998; 4: 551-565Crossref PubMed Scopus (88) Google Scholar), self-association (10Chen T. Damaj B.B. Herrera C. Lasko P. Richard S. Mol. Cell. Biol. 1997; 17: 5707-5718Crossref PubMed Scopus (163) Google Scholar, 18Di Fruscio M. Chen T. Bonyadi S. Lasko P. Richard S. J. Biol. Chem. 1998; 273: 30122-30130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 19Chen T. Richard S. Mol. Cell. Biol. 1998; 18: 4863-4871Crossref PubMed Scopus (105) Google Scholar, 22Zorn A.M. Krieg P.A. Genes Dev. 1997; 11: 2176-2190Crossref PubMed Scopus (73) Google Scholar), heterodimerization (10Chen T. Damaj B.B. Herrera C. Lasko P. Richard S. Mol. Cell. Biol. 1997; 17: 5707-5718Crossref PubMed Scopus (163) Google Scholar, 20Di Fruscio M. Chen T. Richard S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2710-2715Crossref PubMed Scopus (91) Google Scholar, 23Wu J. Zhou L. Tonissen K. Tee R. Artzt K. J. Biol. Chem. 1999; 274: 29202-29210Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), and protein localization (13Chen T. Boisvert F.M. Bazett-Jones D.P. Richard S. Mol. Biol. Cell. 1999; 10: 3015-3033Crossref PubMed Scopus (124) Google Scholar). GSG domain-containing proteins are called STAR proteins forsignal transduction and activators ofRNA metabolism (24Ebersole T.A. Chen Q. Justice M.J. Artzt K. Nat. Genet. 1996; 12: 260-265Crossref PubMed Scopus (287) Google Scholar, 25Vernet C. Artzt K. Trends Genet. 1997; 13: 479-484Abstract Full Text PDF PubMed Scopus (256) Google Scholar), and Sam68 is the prototype because of its links to signaling proteins (5Richard S., Yu, D. Blumer K.J. Hausladen D. Olszowy M.W. Connelly P.A. Shaw A.S. Mol. Cell. Biol. 1995; 15: 186-197Crossref PubMed Google Scholar, 9Taylor S.J. Anafi M. Pawson T. Shalloway D. J. Biol. Chem. 1995; 270: 10120-10124Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The GSG domain is found in a rapidly growing family of RNA-binding proteins (25Vernet C. Artzt K. Trends Genet. 1997; 13: 479-484Abstract Full Text PDF PubMed Scopus (256) Google Scholar). Genetic and biochemical evidence has demonstrated that STAR proteins are involved in many essential processes such as splicing (26Arning S. Gruter P. Bilbe G. Kramer A. RNA (N. Y.). 1996; 2: 794-810PubMed Google Scholar, 27Berglund J.A. Abovich N. Rosbash M. Genes Dev. 1998; 12: 858-867Crossref PubMed Scopus (190) Google Scholar), tumorigenesis (17Jones A.R. Schedl T. Genes Dev. 1995; 9: 1491-1504Crossref PubMed Scopus (220) Google Scholar, 28Liu K. Li L. Nisson P.E. Gruber C. Jessee J. Cohen S.N. J. Biol. Chem. 2000; 275: 40195-40201Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), apoptosis (18Di Fruscio M. Chen T. Bonyadi S. Lasko P. Richard S. J. Biol. Chem. 1998; 273: 30122-30130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 19Chen T. Richard S. Mol. Cell. Biol. 1998; 18: 4863-4871Crossref PubMed Scopus (105) Google Scholar, 29Pilotte J. Larocque D. Richard S. Genes Dev. 2001; 15: 845-858Crossref PubMed Scopus (84) Google Scholar), cell cycle progression (30Barlat I. Maurier F. Duchesne M. Guitard E. Tocque B. Schweighoffer F. J. Biol. Chem. 1997; 272: 3129-3132Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), translation (31Jan E. Motzny C.K. Graves L.E. Goodwin E.B. EMBO J. 1999; 18: 258-269Crossref PubMed Scopus (197) Google Scholar, 32Clifford R. Lee M.H. Nayak S. Ohmachi M. Giorgini F. Schedl T. Development. 2000; 127: 5265-5276PubMed Google Scholar), and development (17Jones A.R. Schedl T. Genes Dev. 1995; 9: 1491-1504Crossref PubMed Scopus (220) Google Scholar, 22Zorn A.M. Krieg P.A. Genes Dev. 1997; 11: 2176-2190Crossref PubMed Scopus (73) Google Scholar, 33Baehrecke E.H. Development. 1997; 124: 1323-1332PubMed Google Scholar, 34Zaffran S. Astier M. Gratecos D. Semeriva M. Development. 1997; 124: 2087-2098Crossref PubMed Google Scholar). The physiological importance of the GSG domain is demonstrated by the fact that many genetic mutations that result in growth or developmental defects have been identified in this protein module. In the nematodeCaenorhabditis elegans, the GSG protein GLD-1 functions as a tumor suppressor that is required for normal oocyte development (35Francis R. Maine E. Schedl T. Genetics. 1995; 139: 607-630Crossref PubMed Google Scholar,36Francis R. Barton M.K. Kimble J. Schedl T. Genetics. 1995; 139: 579-606Crossref PubMed Google Scholar). Thirty-two gld-1 mutations have been identified that fall into six phenotypic classes (17Jones A.R. Schedl T. Genes Dev. 1995; 9: 1491-1504Crossref PubMed Scopus (220) Google Scholar). In mice, a missense mutation in the quaking gene (qk) has been identified (24Ebersole T.A. Chen Q. Justice M.J. Artzt K. Nat. Genet. 1996; 12: 260-265Crossref PubMed Scopus (287) Google Scholar) that is known to be embryonic-lethal (37Bode V.C. Genetics. 1984; 108: 457-470PubMed Google Scholar). This mutation, altering glutamic acid 48 to glycine (24Ebersole T.A. Chen Q. Justice M.J. Artzt K. Nat. Genet. 1996; 12: 260-265Crossref PubMed Scopus (287) Google Scholar), occurs in the NK region of the GSG domain and has been shown to prevent QKI dimerization (19Chen T. Richard S. Mol. Cell. Biol. 1998; 18: 4863-4871Crossref PubMed Scopus (105) Google Scholar). In Drosophila melanogaster, HOW plays a critical role in skeletal muscle development, because weak alleles result in the “held-out-wings” phenotype (33Baehrecke E.H. Development. 1997; 124: 1323-1332PubMed Google Scholar, 34Zaffran S. Astier M. Gratecos D. Semeriva M. Development. 1997; 124: 2087-2098Crossref PubMed Google Scholar). The phenotype of the quaking viable and lethalmice suggests that the QKI proteins are involved in myelination and early embryogenesis (24Ebersole T.A. Chen Q. Justice M.J. Artzt K. Nat. Genet. 1996; 12: 260-265Crossref PubMed Scopus (287) Google Scholar, 38Hogan E.L. Greenfield S. Myelin. Plenum Publishing Corp., New York1984Google Scholar). The mouse qk gene expresses at least five alternatively spliced mRNAs including QKI-5, QKI-6, and QKI-7 that differ in their C-terminal 30 amino acids (24Ebersole T.A. Chen Q. Justice M.J. Artzt K. Nat. Genet. 1996; 12: 260-265Crossref PubMed Scopus (287) Google Scholar). Thequaking viable mutation, which prevents the expression of QKI-6 and QKI-7 isoforms in oligodendrocytes (39Hardy R.J. Loushin C.L. Friedrich V.L. Chen Q. Ebersole T.A. Lazzarini R.A. Artzt K. J. Neurosci. 1996; 16: 7941-7949Crossref PubMed Google Scholar), severely impairs myelination, and as a result the mice develop a characteristic tremor after birth (40Sidman R.L. Dickie M.M. Appel S.H. Science. 1964; 144: 309-311Crossref PubMed Scopus (272) Google Scholar). The specific RNA targets of QKI have not been identified, but the sequence identity between C. elegansGLD-1 and mouse QKI proteins has led Goodwin and co-workers (41Saccomanno L. Loushin C. Jan E. Punkay E. Artzt K. Goodwin E.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12605-12610Crossref PubMed Scopus (96) Google Scholar) to examine whether the introduction of QKI-6 in C. elegans can regulate the specific RNA target of GLD-1. Indeed QKI-6, similar to GLD-1, translationally suppressed the expression of TRA-2 and bound with high affinity to the “tra-2 and GLIelements” (TGEs) (41Saccomanno L. Loushin C. Jan E. Punkay E. Artzt K. Goodwin E.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12605-12610Crossref PubMed Scopus (96) Google Scholar). Previously we have shown that the Sam68 GSG domain in addition to 50 amino acids at its C terminus are necessary and sufficient for RNA binding (10Chen T. Damaj B.B. Herrera C. Lasko P. Richard S. Mol. Cell. Biol. 1997; 17: 5707-5718Crossref PubMed Scopus (163) Google Scholar). To examine the role of the C-terminal amino acids in RNA binding, we generated chimeric proteins between two STAR proteins, Sam68 and QKI. Because the primary amino acid sequence of QKI-5, -6, and -7 isoforms are identical except for the last 6–30 amino acids (depending on the isoform) and are predicted to have identical RNA binding specificity, we chose QKI-7 for our analysis. Here we identified two small regions harboring arginine-glycine repeats in Sam68 that can confer nonspecific RNA binding activity to an adjacent GSG domain. In addition, a novel Sam68 dimerization region has been identified in its C-terminal sequences. The constructs encoding Myc-QKI-7, Myc-QKI-7:E48G, Myc-Sam68, and Myc-Sam68Δ1–67 (now renamed SΔN) were described previously (5Richard S., Yu, D. Blumer K.J. Hausladen D. Olszowy M.W. Connelly P.A. Shaw A.S. Mol. Cell. Biol. 1995; 15: 186-197Crossref PubMed Google Scholar, 10Chen T. Damaj B.B. Herrera C. Lasko P. Richard S. Mol. Cell. Biol. 1997; 17: 5707-5718Crossref PubMed Scopus (163) Google Scholar, 19Chen T. Richard S. Mol. Cell. Biol. 1998; 18: 4863-4871Crossref PubMed Scopus (105) Google Scholar). Myc-S-Q encodes a chimeric protein containing the N-terminal region of Sam68 and the C-terminal region of QKI-7. The DNA sequence encoding the C-terminal 180 amino acids of Myc-Sam68Δ1–67 was removed by restriction endonuclease digestion using EcoRV and KpnI and replaced with a sequence encoding the C-terminal 136 amino acids of QKI-7. The QKI-7 fragment was generated with polymerase chain reaction (PCR) using Myc-QKI-7 as the DNA template and the universal reverse primer and 5′-GAC GAT ATC AAG AAG ATG CAG CTG ATG-3′ (theEcoRV site is underlined) as oligonucleotides. The chimeric protein Myc-Q-S contains the N-terminal region of QKI-7 and the C-terminal region of Sam68. The plasmid expressing this protein was constructed as follows: a DNA fragment encoding the C-terminal 188 amino acids of Sam68 was generated with PCR using Myc-Sam68 as the DNA template, 5′-CCT GGT ACC AGA TAT GAT GGA T-3′ as the forward primer, and universal reverse primer as the reverse primer, the fragment was digested with KpnI (the restriction site is underlined) and subcloned into Myc-QKI-7, the C-terminal 145 amino acids of which had been removed with KpnI digestion. Myc-Q-S:E→G was constructed using the same strategy as that of Myc-Q-S except that Myc-QKI-7:E48G, instead of Myc-QKI-7, was used to subclone the Sam68 DNA fragment. The construct encoding Myc-Q-S:A→N was generated by inverse PCR using Myc-Q-S as the DNA template and 5′-ATT CAA CTT GAA GCA GAA ACG GGA-3′ and 5′-ATT TGT AAG TCC TCT AGG TCC AAG-3′ as primers (underlined nucleotides denote changes introduced). The Myc-Q-S and Myc-S-Q C-terminal deletion constructs, Myc-Q-S330, Myc-Q-S294, Myc-S-Q284, and Myc-S-Q205, were generated with PCR using Myc-Q-S and Myc-S-Q as the DNA templates, respectively. The T7 promoter primer was used as the forward primer and the following oligonucleotides were used as reverse primers: 5′-AGG AAT TCA TGG CAC CCC TCG AGT CAC A-3′ (for Myc-Q-S330), 5′-TAG AAT TCA GGC AGC TCC TCG TCC TCT CAC-3′ (for Myc-Q-S294), 5′-AGG GAA TTC AGA TTA ACC CAG CTT CAG GCC-3′ (for Myc-S-Q284), and 5′-ATG GAA TTC TAT CTG TAG GTG CCA TTC AG-3′ (for Myc-S-Q205). The amplified DNA fragments were digested with EcoRI and subcloned into Myc-Bluescript KS(+) (5Richard S., Yu, D. Blumer K.J. Hausladen D. Olszowy M.W. Connelly P.A. Shaw A.S. Mol. Cell. Biol. 1995; 15: 186-197Crossref PubMed Google Scholar, 10Chen T. Damaj B.B. Herrera C. Lasko P. Richard S. Mol. Cell. Biol. 1997; 17: 5707-5718Crossref PubMed Scopus (163) Google Scholar). The plasmids encoding Myc-Q(GSG)-S, Myc-Q(GSG)-SΔ4RG, and Myc-Q(GSG)-SΔ6RG were constructed by a two-step subcloning strategy. DNA fragments encoding the C-terminal regions of Sam68 were amplified by PCR with universal reverse primer and 5′-GTG GTC GAC GGG TAT CTG TGA GAG GAC-3′ for Myc-Q(GSG)-S, 5′-GGA GTC GAC CCT CCT CCT CCA CCT GT-3′ for Myc-Q(GSG)-SΔ4RG or 5′-GTG GTC GAC CAC CTA GAG GAG CTT-3′ for Myc-Q(GSG)-SΔ6RG as primers. The fragments were digested with SalI (the restriction site is underlined) and KpnI (the site is in the vector) and inserted in the same sites of pBluescript KS(+). The resulting plasmids were then used to subclone the DNA fragments encoding Myc-tagged N-terminal regions of QKI-7. The Myc-QKI-7 fragments were generated by PCR using Myc-QKI-7 as template, T7 promoter primer as the forward primer, and the following oligonucleotides as reverse primers: 5′-CTG GTC GAC TAA TGT TGG CGT CTC TGT-3′ for Myc-Q(GSG)-S, 5′-AGT GTC GAC AAG AGA AAA GGC AAG GGC-3′ for Myc-Q(GSG)-SΔ4RG, and 5′-CAG GTC GAC GCC CAG TGA TGA TCC TTG-3′ for Myc-Q(GSG)-SΔ6RG. These fragments were digested with BamHI (the site is in the vector) and SalI (the site is underlined) and subcloned in the corresponding Sam68-pBluescript plasmids described above. The construct encoding Myc-Q(GSG)-SΔ11RG was also generated in two steps. A PCR fragment encoding the Myc-epitope tag and the N-terminal 256 amino acids of QKI-7 was first subcloned in the BamHI andSalI sites of pBluescript KS(+), and the XhoI fragment (C-terminal 112 amino acids) of Sam68 was then inserted in theSalI site of the resulting plasmid. To generate the Myc-QKI:1–256 fragment, Myc-QKI-7 was used as DNA template and T7 promoter primer and 5′-GTA GTC GAC TGA TCA AAG GCA TTA-3′ as primers (the SalI site is underlined). Myc-QKI-5RG is a chimeric protein in which a Sam68 sequence harboring five RG repeats is introduced in QKI-7. The plasmid encoding this protein was generated as follows: a PCR fragment encoding the C-terminal 65 amino acids of QKI-7 was first inserted in the HindIII and KpnI sites of pBluescript KS(+), and a second PCR fragment containing coding sequences for the Myc tag, QKI-7 amino acids 1–231 and Sam68 amino acids 308–333, was then subcloned in the BamHI andHindIII sites of the resulting plasmid. The first PCR fragment was generated using Myc-QKI-7 as template and universal reverse primer and 5′-AGA AAG CTT TCA TGC CAA ACG GAA CTC-3′ as primers. The second PCR fragment was generated using Myc-Q(GSG)-SΔ6RG as template and T7 promoter primer and 5′-GTGAAG CTT GCA CCC CTC GAG TCA CAG-3′ as primers (theHindIII site is underlined). The constructs encoding Myc-SΔN:315RG→AS, Myc-SΔN:320RG→AS, Myc-SΔN:325RG→AS, Myc-SΔN:315,325RG→AS, Myc-SΔN:Δ3RG, Myc-Sam68:Δ3RG, Myc-Sam68R→A:10,13,17, Myc-Sam68R→A:43,45, and Myc-Sam68R→A:52,56 were all generated by inverse PCR. The DNA templates used were Myc-SΔN (for Myc-SΔN:Δ3RG and -SΔN:RG→AS constructs except Myc-SΔN:315,325RG→AS), Myc-SΔN:315RG→AS (for Myc-SΔN:315,325RG→AS), Myc-Sam68 (for Myc-Sam68:Δ3RG), and Myc-Sam68:Δ3RG (for Myc-Sam68R→A constructs), and the primer pairs were: 5′-AGCACC CCA GTG AGA GGC TCC-3′ and 5′-AGC AAC CAA AGC TCC TCT AGG-3′ (for Myc-SΔN:315RG→AS), 5′-AGC TCC ATC ACC AGA GGT G-3′ and 5′-AGC CAC TGG GGT TCC ACG AAC-3′ (for Myc-SΔN:320RG→AS), 5′-AGC GCC ACT GTG ACT CGA GGG-3′ and 5′-AGC GGT GAT GGA GCC TCT CAC-3′ (for Myc-SΔN:325RG→AS and -SΔN:315,325RG→AS), 5′-AGC GCC ACT GTG ACT CGA GGG-3′ and 5′-AGC AAC CAA AGC TCC TCT AGG-3′ (for Myc-SΔN:Δ3RG and -Sam68:Δ3RG), 5′-AGC TCG GGC GCC AGC TGC TCC AAG GAC CCG-3′ and 5′-AGC GGT GAG GGC CGA GGC AGG ATC GTC CCG-3′ (for Myc-Sam68R→A:10, 13, 17), 5′-AGC GGG GGA GGT GGG CCC AGA-3′ and 5′-AGC GGG CGC GTG AGG AAG CGG CGA CGG-3′ (for Myc-Sam68R→A:43,45), and 5′-AGC GGC GCTGCG GCC TCG CCCGCC ACC CAG-3′ and 5′-AGC GGG CCC ACC TCC CCC TCC-3′ (for Myc-Sam68R→A:52,56). Underlined nucleotides denote changes introduced. The construct encoding the G8–5 RNA sequence (12Lin Q. Taylor S.J. Shalloway D. J. Biol. Chem. 1997; 272: 27274-27280Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), 5′-GGG UGA CAC ACU AGC UAU AGC AUU AAA AGA CCG AGC AAG U-3′ (the UAAA motif is underlined), was generated by annealing two oligonucleotides (5′-GCCGAA TTC GGG TGA CAC ACT AGC TAT AGC ATT A-3′ and 5′-AGCTCT AGA CTT GCT CGG TCT TTT AAT GCT ATA GCT-3′) and filling in the ends with the Klenow fragment of DNA polymerase I. This DNA fragment was digested with EcoRI and XbaI (the restriction sites are underlined) and subcloned into pBluescript SK(+). The plasmids encoding the tra-2 3′ UTR and a deletion mutant of tra-2 3′ UTR, (−108) 3′ UTR, were kindly provided by Tim Schedl (Washington University). The identities of all the above plasmid constructs were verified by dideoxynucleotide sequencing. Proteins were expressed in HeLa cells, using the vaccinia virus T7 expression system as described previously (5Richard S., Yu, D. Blumer K.J. Hausladen D. Olszowy M.W. Connelly P.A. Shaw A.S. Mol. Cell. Biol. 1995; 15: 186-197Crossref PubMed Google Scholar). HeLa cells were lysed in lysis buffer (1% Triton X-100, 150 mm NaCl, and 20 mm Tris-HCl (pH 8.0)), 50 mm NaF, 100 mm sodium vanadate, 0.01% phenylmethanesulfonyl fluoride, 1 µg of aprotinin/ml, and 1 µg of leupeptin/ml), and the cellular debris and nuclei were removed by centrifugation. For immunoprecipitation, the supernatant was incubated on ice with the specified antibody for 1 h. Then 20 µl of a 50% protein A-Sepharose slurry was added and incubated at 4 °C for 30 min with constant end-over-end mixing. The beads were washed twice with lysis buffer and once with PBS. Protein samples were analyzed on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Immunoblotting was performed using the anti-Myc (9E10), anti-hemagglutinin (HA), or anti-p59fyn antibodies. The rabbit anti-p59fyn antibody was provided kindly by AndréVeillette (Institut de Recherche Clinique de Montréal, Université de Montréal, McGill University). The designated primary antibody was followed by goat anti-mouse or goat anti-rabbit antibodies conjugated to horse radish peroxidase (ICN), and chemiluminescence was used for protein detection (DuPont). 32P-labeled G8–5 RNA and tra-2 3′-UTR RNA were transcribed in vitrowith the T7 RNA polymerase following the protocols recommended by the manufacturer (Promega). After in vitro transcription, the template DNA was digested with DNase I (Promega), and the RNA was extracted with phenol-chloroform, precipitated with ethanol, and resuspended in diethyl pyrocarbonate-treated water at a concentration of 106 cpm/µl. For the poly(U) binding assay, Myc-tagged proteins expressed in HeLa cells were incubated at 4 °C for 30 min with poly(U)-Sepharose beads or control Sepharose beads in lysis buffer supplemented with 2 mg/ml heparin. The bound proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-Myc antibodies. For G8–5 or tra-23′-UTR RNA binding, Myc-tagged proteins expressed in HeLa cells were immunoprecipitated with an anti-Myc antibody or mouse IgG (control), and the immunoprecipitates were incubated at 4 °C for 30 min with 1 µl (106 cpm) of 32P-labeled RNA in lysis buffer supplemented with 2 mg/ml heparin. The beads were washed twice with lysis buffer and once with PBS, and the bound radioactivity was quantitated by scintillation counting. To verify the identity of the radiolabeled RNA bound to beads, the bound RNA was eluted with sample buffer and analyzed with nondenaturing polyacrylamide gel electrophoresis and autoradiography. To verify protein expression, the immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody. REV assays were performed as described previously (7Derry J.J. Richard S. Valderrama Carvajal H. Ye X. Vasioukhin V. Cochrane A.W. Chen T. Tyner A.L. Mol. Cell. Biol. 2000; 20: 6114-6126Crossref PubMed Scopus (134) Google Scholar). STAR proteins contain a GSG domain, which is a tripartite protein module containing from N to C terminus the NK region, the KH domain, and the CK region (Fig. 1 B). The Sam68 KH domain is necessary for RNA binding because its deletion prevents RNA binding (10Chen T. Damaj B.B. Herrera C. Lasko P. Richard S. Mol. Cell. Biol. 1997; 17: 5707-5718Crossref PubMed Scopus (163) Google Scholar, 12Lin Q. Taylor S.J. Shalloway D. J. Biol. Chem. 1997; 272: 27274-27280Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 30Barlat I. Maurier F. Duchesne M. Guitard E. Tocque B. Schweighoffer F. J. Biol. Chem. 1997; 272: 3129-3132Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). To investigate whether the RNA binding specificity of the STAR proteins resides only in the KH domain or whether neighboring regions can regulate RNA binding, we constructed chimeric proteins between QKI-7 and Sam68. These two proteins show a high degree of homology in their GSG domain (Fig. 1 A) but possess distinct RNA binding specificities. Sam68 has been shown to bind homopolymeric RNA poly(U) and poly(A) (2Taylor S.J. Shalloway D. Nature. 1994; 368: 867-871Crossref PubMed Scopus (373) Google Scholar, 10Chen T. Damaj B.B. Herrera C. Lasko P. Richard S. Mol. Cell. Biol. 1997; 17: 5707-5718Crossref PubMed Scopus (163) Google Scholar) as well as a synthetic RNA (G8–5) amplified by using systematic evolution of ligands by exponential enrichment (12Lin Q. Taylor S.J. Shalloway D. J. Biol. Chem. 1997; 272: 27274-27280Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The QKI proteins bindC. elegans GLD-1 target, tra-2 (41Saccomanno L. Loushin C. Jan E. Punkay E. Artzt K. Goodwin E.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12605-12610Crossref PubMed Scopus (96) Google Scholar), but not homopolymeric RNA (10Chen T. Damaj B.B. Herrera C. Lasko P. Richard S. Mol. Cell. Biol. 1997; 17: 5707-5718Crossref PubMed Scopus (163) Google Scholar). Chimeric proteins were generated between Sam68 and QKI-7 in such a way that the CK region and the C terminus of one protein was replaced by the corresponding region of the other protein (Fig. 1 B). Q-S (QKI-7-Sam68 chimeric protein) contains the NK region and the KH domain of QKI-7 and the CK region and the C terminus of Sam68 (Fig. 1 B
Heterogeneous ribonucleoprotein particle
RNA recognition motif
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Conformational change
Dissociation constant
Site-directed mutagenesis
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