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• W2000329656 abstract "Our previous work identified E3 ubiquitin ligases, termed UBR1-UBR7, that contain the ∼70-residue UBR box, a motif important for the targeting of N-end rule substrates. In this pathway, specific N-terminal residues of substrates are recognized as degradation signals by UBR box-containing E3s that include UBR1, UBR2, UBR4, and UBR5. The other E3s of this set, UBR3, UBR6, and UBR7, remained uncharacterized. Here we describe the cloning and analyses of mouse UBR3. The similarities of UBR3 to the UBR1 and UBR2 E3s of the N-end rule pathway include the RING and UBR domains. We show that HR6A and HR6B, the E2 enzymes that bind to UBR1 and UBR2, also interact with UBR3. However, in contrast to UBR1 and UBR2, UBR3 does not recognize N-end rule substrates. We also constructed UBR3-lacking mouse strains. In the 129SvImJ background, UBR3-/- mice died during embryogenesis, whereas the C57BL/6 background UBR3-/- mice exhibited neonatal lethality and suckling impairment that could be partially rescued by litter size reduction. The adult UBR3-/- mice had female-specific behavioral anosmia. Cells of the olfactory pathway were found to express β-galactosidase (LacZ) that marked the deletion/disruption UBR3- allele. The UBR3-specific LacZ expression was also prominent in cells of the touch, vision, hearing, and taste systems, suggesting a regulatory role of UBR3 in sensory pathways, including olfaction. By analogy with functions of the UBR domain in the N-end rule pathway, we propose that the UBR box of UBR3 may recognize small compounds that modulate the targeting, by this E3, of its currently unknown substrates. Our previous work identified E3 ubiquitin ligases, termed UBR1-UBR7, that contain the ∼70-residue UBR box, a motif important for the targeting of N-end rule substrates. In this pathway, specific N-terminal residues of substrates are recognized as degradation signals by UBR box-containing E3s that include UBR1, UBR2, UBR4, and UBR5. The other E3s of this set, UBR3, UBR6, and UBR7, remained uncharacterized. Here we describe the cloning and analyses of mouse UBR3. The similarities of UBR3 to the UBR1 and UBR2 E3s of the N-end rule pathway include the RING and UBR domains. We show that HR6A and HR6B, the E2 enzymes that bind to UBR1 and UBR2, also interact with UBR3. However, in contrast to UBR1 and UBR2, UBR3 does not recognize N-end rule substrates. We also constructed UBR3-lacking mouse strains. In the 129SvImJ background, UBR3-/- mice died during embryogenesis, whereas the C57BL/6 background UBR3-/- mice exhibited neonatal lethality and suckling impairment that could be partially rescued by litter size reduction. The adult UBR3-/- mice had female-specific behavioral anosmia. Cells of the olfactory pathway were found to express β-galactosidase (LacZ) that marked the deletion/disruption UBR3- allele. The UBR3-specific LacZ expression was also prominent in cells of the touch, vision, hearing, and taste systems, suggesting a regulatory role of UBR3 in sensory pathways, including olfaction. By analogy with functions of the UBR domain in the N-end rule pathway, we propose that the UBR box of UBR3 may recognize small compounds that modulate the targeting, by this E3, of its currently unknown substrates. A protein substrate of the ubiquitin (Ub) 2The abbreviations used are: Ub, ubiquitin; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; GPCR, G protein-coupled receptor; GST, glutathione S-transferase; MOE, main olfactory epithelium; VNO, vomeronasal organ; OB, olfactory bulb; OC, olfactory cortex; HPLC, high performance liquid chromatography; ES, embryonic stem. -proteasome system, which controls the levels of many intracellular proteins, is conjugated to Ub through the action of E1, E2, and E3 enzymes (1Fang S. Weissman A.M. Cell Mol. Life. Sci. 2004; 61: 1546-1561Crossref PubMed Google Scholar, 2Hochstrasser M. Cell. 2006; 124: 27-34Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 3Petroski M.D. Deshaies R.J. Nat. Rev. Mol. Cell. Biol. 2005; 6: 9-20Crossref PubMed Scopus (1682) Google Scholar, 4Varshavsky A. Pro. Sci. 2006; 15: 647-654Crossref PubMed Scopus (58) Google Scholar). The selectivity of ubiquitylation is mediated largely by E3, which recognizes a substrate's degradation signal (degron) (5Bachmair A. Varshavsky A. Cell. 1989; 56: 1019-1032Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 6Gardner R.G. Hampton R.Y. EMBO J. 1999; 18: 5994-6004Crossref PubMed Scopus (75) Google Scholar, 7Suzuki T. Varshavsky A. EMBO J. 1999; 18: 6017-6026Crossref PubMed Scopus (94) Google Scholar, 8Sheng J. Kumagai A. Dunphy W.G. Varshavsky A. EMBO J. 2002; 21: 6061-6071Crossref PubMed Scopus (39) Google Scholar). The E3 Ub ligases are an exceptionally large family, with more than 500 distinct E3s in a mammal (9Cardozo T. Pagano M. Nat. Rev. Mol. Cell. Biol. 2004; 5: 739-751Crossref PubMed Scopus (884) Google Scholar, 10Ardley H.C. Robinson P.A. Essays Biochem. 2005; 41: 15-30Crossref PubMed Google Scholar). The term “Ub ligase” denotes either an E2-E3 holoenzyme or its E3 component. A ubiquitylated protein bears a covalently linked poly-Ub chain and is targeted for processive degradation by the 26 S proteasome (11Baumeister W. Walz J. Zühl F. Seemüller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1306) Google Scholar). Ub has other functions as well, including non-proteolytic ones (12Finley D. Bartel B. Varshavsky A. Nature. 1989; 338: 394-401Crossref PubMed Scopus (557) Google Scholar, 13Schnell J.D. Hicke L. J. Biol. Chem. 2003; 278: 35857-35860Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). An essential determinant of one class of degrons, called N-degrons, is a substrate's destabilizing N-terminal residue. The set of destabilizing residues in a given cell type yields a rule, called the N-end rule, which relates the in vivo half-life of a protein to the identity of its N-terminal residue (Fig. 1A) (4Varshavsky A. Pro. Sci. 2006; 15: 647-654Crossref PubMed Scopus (58) Google Scholar, 14Bachmair A. Finley D. Varshavsky A. Science. 1986; 234: 179-186Crossref PubMed Scopus (1383) Google Scholar, 15Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (721) Google Scholar, 16Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (123) Google Scholar, 17Tasaki T. Mulder L.C.F. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (245) Google Scholar, 18Hu R.-G. Sheng J. Xin Q. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (239) Google Scholar, 19Hu R.-G. Brower C.S. Wang H. Davydov I.V. Sheng J. Zhou J. Kwon Y.T. Varshavsky A. J. Biol. Chem. 2006; 281: 32559-32573Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In eukaryotes, the N-degron consists of three determinants: a destabilizing N-terminal residue of a protein substrate, its internal Lys residue(s) (the site of formation of a poly-Ub chain), and a conformationally flexible region(s) in the vicinity of these determinants (5Bachmair A. Varshavsky A. Cell. 1989; 56: 1019-1032Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 7Suzuki T. Varshavsky A. EMBO J. 1999; 18: 6017-6026Crossref PubMed Scopus (94) Google Scholar, 15Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (721) Google Scholar, 20Johnson E.S. Gonda D.K. Varshavsky A. Nature. 1990; 346: 287-291Crossref PubMed Scopus (108) Google Scholar, 21Prakash S. Tian L. Ratliff K.S. Lehotzky R.E. Matouschek A. Nat. Struct. Mol. Biol. 2004; 11: 830-837Crossref PubMed Scopus (360) Google Scholar). The N-end rule has a hierarchic structure in that some of the destabilizing N-terminal residues (Arg, Lys, His, Phe, Leu, Trp, Tyr, and Ile) are recognized directly by E3 Ub ligases of the N-end rule pathway, called N-recognins, whereas the other destabilizing N-terminal residues (Asn, Gln, Asp, Glu, and Cys) must be modified in vivo, enzymatically or otherwise, for their subsequent (indirect) recognition by these E3s (Fig. 1A) (18Hu R.-G. Sheng J. Xin Q. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (239) Google Scholar, 19Hu R.-G. Brower C.S. Wang H. Davydov I.V. Sheng J. Zhou J. Kwon Y.T. Varshavsky A. J. Biol. Chem. 2006; 281: 32559-32573Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 22Kwon Y.T. Balogh S.A. Davydov I.V. Kashina A.S. Yoon J.K. Xie Y. Gaur A. Hyde L. Denenberg V.H. Varshavsky A. Mol. Cell. Biol. 2000; 20: 4135-4148Crossref PubMed Scopus (88) Google Scholar, 23Kwon Y.T. Kashina A.S. Varshavsky A. Mol. Cell. Biol. 1999; 19: 182-193Crossref PubMed Scopus (110) Google Scholar, 24Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.-G. An J.Y. Seo J.W. Du F. Varshavsky A. Science. 2002; 297: 96-99Crossref PubMed Scopus (262) Google Scholar, 25Lee M.J. Tasaki T. Moroi K. An J.Y. Kimura S. Davydov I.V. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15030-15035Crossref PubMed Scopus (191) Google Scholar). The functions of the N-end rule pathway include its roles in the control of signaling by nitric oxide and G protein-coupled receptors (GPCRs), the regulation of peptide import, the fidelity of chromosome segregation, the apoptosis, meiosis, cardiovascular development, neurogenesis, and pancreatic functions, and leaf senescence in plants (16Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (123) Google Scholar, 18Hu R.-G. Sheng J. Xin Q. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (239) Google Scholar, 24Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.-G. An J.Y. Seo J.W. Du F. Varshavsky A. Science. 2002; 297: 96-99Crossref PubMed Scopus (262) Google Scholar, 25Lee M.J. Tasaki T. Moroi K. An J.Y. Kimura S. Davydov I.V. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15030-15035Crossref PubMed Scopus (191) Google Scholar, 26Turner G.C. Du F. Varshavsky A. Nature. 2000; 405: 579-583Crossref PubMed Scopus (165) Google Scholar, 27Rao H. Uhlmann F. Nasmyth K. Varshavsky A. Nature. 2001; 410: 955-960Crossref PubMed Scopus (230) Google Scholar, 28Ditzel M. Wilson R. Tenev T. Zachariou A. Paul A. Deas E. Meier P. Nat. Cell Biol. 2003; 5: 467-473Crossref PubMed Scopus (206) Google Scholar, 29Varshavsky A. Nat. Cell Biol. 2003; 5: 373-376Crossref PubMed Scopus (93) Google Scholar, 30Yoshida S. Ito M. Gallis J. Nishida I. Watanabe A. Plant J. 2002; 32: 129-137Crossref PubMed Scopus (120) Google Scholar, 31An J.Y. Seo J.W. Tasaki T. Lee M.J. Varshavsky A. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6212-6217Crossref PubMed Scopus (73) Google Scholar, 32Zenker M. Mayerle J. Lerch M.M. Tagariello A. Zerres K. Durie P.R. Beier M. Hülskamp G. Guzman C. Rehder H. Beemer F.A. Hamel B. Vanlieferinghen P. Gershoni-Baruch R. Vieira M.W. Dumic M. Auslender R. Gil-da-Silva-Lopes V.L. Steinlicht S. Rauh R. Shalev S.A. Thiel C. Winterpacht A. Kwon Y.T. Varshavsky A. Reis A. Nat. Genet. 2005; 37: 1345-1350Crossref PubMed Scopus (193) Google Scholar). The N-end rule pathway of the yeast Saccharomyces cerevisiae is mediated by a single N-recognin, the Ub ligase UBR1 (33Du F. Navarro-Garcia F. Xia Z. Tasaki T. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14110-14115Crossref PubMed Scopus (84) Google Scholar), whereas at least four N-recognins, including UBR1, mediate this pathway in mammals (16Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (123) Google Scholar, 17Tasaki T. Mulder L.C.F. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (245) Google Scholar, 31An J.Y. Seo J.W. Tasaki T. Lee M.J. Varshavsky A. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6212-6217Crossref PubMed Scopus (73) Google Scholar, 34Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (116) Google Scholar). Our recent study (17Tasaki T. Mulder L.C.F. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (245) Google Scholar) identified a family of mammalian E3 Ub ligases that share a characteristic zinc finger-like ∼70-residue domain termed the UBR box. The mouse genome encodes at least seven UBR box-containing proteins, termed UBR1-UBR7, which vary both in size (from 50 to 570 kDa) and in containing or lacking other E3-characteristic domains, such as HECT, RING, PHD, or F-box (Fig. 1B) (17Tasaki T. Mulder L.C.F. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (245) Google Scholar). UBR1 and UBR2 of this set, the highly homologous (47% identical) 200-kDa E3s, are the known N-recognins of the mouse N-end rule pathway (16Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (123) Google Scholar, 34Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (116) Google Scholar). In the S. cerevisiae RING-type UBR1 Ub ligase and also (by inference) in its mammalian homologs UBR1 and UBR2, the UBR box and nearby regions mediate the recognition of N-degrons (i.e. the binding of N-recognin to a primary destabilizing N-terminal residue (Arg, Lys, His, Phe, Leu, Trp, Tyr, or Ile) of a polypeptide) (16Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (123) Google Scholar, 33Du F. Navarro-Garcia F. Xia Z. Tasaki T. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14110-14115Crossref PubMed Scopus (84) Google Scholar). S. cerevisiae UBR1 recognizes type-1 (basic) destabilizing N-terminal residues Arg, Lys, and His through a binding site that resides at least in part in the UBR box. UBR1 recognizes type-2 (bulky hydrophobic) N-terminal residues Phe, Leu, Trp, Tyr, and Ile through a nearby but genetically distinguishable domain that includes the UBR box and a proximal downstream region (see Refs. 17Tasaki T. Mulder L.C.F. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (245) Google Scholar, 33Du F. Navarro-Garcia F. Xia Z. Tasaki T. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14110-14115Crossref PubMed Scopus (84) Google Scholar, and 35Erbse A. Schmidt R. Bornemann T. Schneider-Mergener J. Mogk A. Zahn R. Dougan D.A. Bukau B. Nature. 2005; 439: 753-756Crossref Scopus (182) Google Scholar and references therein). S. cerevisiae UBR1 contains yet another (third) substrate-binding site. Its activity is allosterically controlled by occupancy states of the other two (type-1 and type-2) binding sites of UBR1. The third substrate-binding site targets the transcriptional repressor CUP9, through an internal degron near the C terminus of CUP9 (26Turner G.C. Du F. Varshavsky A. Nature. 2000; 405: 579-583Crossref PubMed Scopus (165) Google Scholar, 33Du F. Navarro-Garcia F. Xia Z. Tasaki T. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14110-14115Crossref PubMed Scopus (84) Google Scholar). Since mouse UBR1 and UBR2 are homologs of S. cerevisiae UBR1 (16Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (123) Google Scholar, 36Kwon Y.T. Reiss Y. Fried V.A. Hershko A. Yoon J.K. Gonda D.K. Sangan P. Copeland N.G. Jenkins N.A. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7898-7903Crossref PubMed Scopus (151) Google Scholar), one would expect these mammalian Ub ligases to also contain analogous (third) substrate-binding sites (Fig. 1A). Indeed, mouse UBR1 was shown to target c-FOS, a component of the AP-1 transcription factor, for ubiquitylation and subsequent degradation through a conditional (regulated by phosphorylation) degron near the N terminus of c-FOS (37Sasaki T. Kojima H. Kishimoto R. Ikeda A. Kunimoto H. Nakajima K. Mol. Cell. 2006; 24: 63-75Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Several other UBR box-containing proteins, such as UBR4 (PUSHOVER/BIG) and UBR5 (EDD/hHYD), are also, operationally, N-recognins whose substrate specificities include the ability to recognize primary destabilizing N-terminal residues (Fig. 1A) (17Tasaki T. Mulder L.C.F. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (245) Google Scholar). We have previously characterized mammalian UBR1, UBR2, UBR4, and UBR5 as Ub ligases of the N-end rule pathway that recognize destabilizing N-terminal residues (16Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (123) Google Scholar, 17Tasaki T. Mulder L.C.F. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (245) Google Scholar, 34Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (116) Google Scholar, 36Kwon Y.T. Reiss Y. Fried V.A. Hershko A. Yoon J.K. Gonda D.K. Sangan P. Copeland N.G. Jenkins N.A. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7898-7903Crossref PubMed Scopus (151) Google Scholar). In the present study, we cloned and characterized mouse UBR3, a protein of unknown function that contains both the UBR box and the RING domain. Our findings show that UBR1, UBR2, and UBR3 are structurally related members of the RING-UBR subfamily of UBR-box E3s that interact (and function together) with the E2 enzymes HR6A and HR6B. We also constructed and characterized mouse strains lacking UBR3 and describe several lines of evidence suggesting that one function of UBR3 involves the regulation of sensory path-ways, including olfaction. Cloning of the UBR3 cDNA—A 412-bp cDNA fragment that encompassed a 3′-proximal region of the UBR3 ORF (see “Results”) was used as a probe to screen a λEXlox-based embryonic day 10 (E10) mouse embryo cDNA library (Novagen, Madison, WI), yielding a 2.5-kb UBR3 cDNA fragment. This fragment was employed as a probe in a second screen, using a λgt10-based mouse cDNA library from MEL-C19 cells (Clontech) and yielding in a 1-kb fragment. Two subsequent screenings yielded 2-kb and 1.5-kb fragments. To isolate the 5′ region of UBR3 cDNA, 5′-rapid amplification of cDNA ends PCR was performed with poly(A)+ RNA from mouse liver or skeletal muscle. The resulting set of overlapping cDNA fragments were joined to produce full-length UBR3 cDNAs encoding two splicing-derived isoforms. Construction of UBR3-/- Mouse Strains—The mouse UBR3 gene was isolated by screening a bacterial artificial chromosome (BAC) DNA library (Genome Systems, St. Louis, MO) from 129SvJ mouse embryonic stem (ES) cells by using a UBR3-specific cDNA probe (nucleotides 385-1,886). The exon/intron organization of the first ∼30 kb of UBR3 was determined using exon-specific PCR primers to amplify genomic DNA fragments flanked by exons as previously described (34Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (116) Google Scholar). The targeting vector was constructed using standard techniques, linearized with KpnI, and electroporated into CJ7 ES cells, followed by standard procedures for selection and identification of correctly targeted UBR3+/- ES cell colonies by using PCR and Southern hybridization (16Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (123) Google Scholar, 22Kwon Y.T. Balogh S.A. Davydov I.V. Kashina A.S. Yoon J.K. Xie Y. Gaur A. Hyde L. Denenberg V.H. Varshavsky A. Mol. Cell. Biol. 2000; 20: 4135-4148Crossref PubMed Scopus (88) Google Scholar, 24Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.-G. An J.Y. Seo J.W. Du F. Varshavsky A. Science. 2002; 297: 96-99Crossref PubMed Scopus (262) Google Scholar, 34Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (116) Google Scholar, 38Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Smith J.A. Seidman J.G. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York2002Google Scholar). The resulting UBR3+/- ES cell clones (6 of 960 ES cell clones analyzed) were further selected for apparently normal karyotypes. Four independently produced UBR3+/- ES cell clones were injected into C57/BL6J blastocysts to generate chimeric mice. In two of these mice, the mutant (UBR3-) allele was transmitted to the germ line. Northern and Southern hybridizations and PCR-mediated genotyping were carried out as described (34Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (116) Google Scholar). GST Pull-down Assays—Glutathione S-transferase (GST) pull-down assays were carried out as described (16Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (123) Google Scholar). N-terminally FLAG-tagged mouse fUBR1, fUBR2, and fUBR3 and S. cerevisiae fUBR1 (sc-fUBR1) were expressed in S. cerevisiae SC295 (MATa, GAL4 GAL80 ura3-52, leu2-3,112 reg1-501 gal1 pep4-3) from the PADH1 promoter in a high copy vector. X-SCC1-GST fusions (where X represents Met, Arg, or Leu) were produced using the IMPACT (intein-mediated purification with an affinity chitin-binding tag) system (New England Biolabs, Ipswich, MA) as previously described (16Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (123) Google Scholar). N-terminal residues of the test proteins were verified by Edman sequencing of purified X-SCC1-GST fusions. GST fusions to mouse HR6A and HR6B (GST-HR6A and GST-HR6B) were expressed in Escherichia coli BL21(DE3) and purified as described (16Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (123) Google Scholar). The purified X-SCC1-GST (2 μg) in 250 μl of the loading buffer (137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, 1.4 mm KH2PO4, 10% glycerol, and 1% Triton X-100, pH 7.4) was incubated with 15 μl of glutathione-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C. The beads were washed once with the loading buffer and twice with the binding buffer (10% glycerol, 0.1% Nonidet P-40, 0.2 m KCl, 1 mm dithiothreitol, and 50 mm HEPES, pH 7.5). The diluted yeast extracts (250 μl) containing E3s in binding buffer containing the protease inhibitor tablet (Roche Applied Science) were added into the washed beads. The bound proteins were eluted, fractionated by SDS-PAGE, and immunoblotted with anti-FLAG antibody. The 5% input lanes (see Fig. 2) refer to a directly loaded sample of the yeast extract that corresponded to 5% of the extract amount used in GST pull-down assays. X-Peptide Pull-down Assay—X-Peptides (X-Ile-Phe-Ser-Thr-Asp-Thr-Gly-Pro-Gly-Gly-Cys, where X represents Arg, Phe, Gly, Ser, Thr, Ala, Asp, or Met), where residues 2-9 were derived from residues 2-9 of Sindbis virus polymerase nsP4, were synthesized as described (17Tasaki T. Mulder L.C.F. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (245) Google Scholar). S. cerevisiae extract expressing mouse fUBR1, fUBR2, or fUBR3 was diluted by the lysis buffer (10% glycerol, 0.1% Nonidet P-40, 0.2 m KCl, 1 mm dithiothreitol, and 50 mm HEPES, pH 7.5) to 1.2 mg/ml of total protein and thereafter incubated with or without an added dipeptide for 15 min on ice to prevent proteolytic degradation of peptide substrates. A sample (0.3 ml) was then transferred to a new tube containing 5 μl (packed volume) of a carrier-linked 12-mer peptide, followed by gentle mixing, through rotation of the tube, for 1 h at 4 °C. The beads were pelleted by a brief centrifugation and then washed three times, for 2 min each, with the lysis buffer. The beads were then suspended in 20 μl of SDS-PAGE loading buffer and heated at 95 °C for 5 min, followed by a brief spin in a microcentrifuge, SDS-8% PAGE of the supernatant, and detection of fUBR1, fUBR2, or fUBR3 by immunoblotting with anti-FLAG M2 antibody. Antibody to Mouse UBR3—An affinity-purified polyclonal rabbit antibody to mouse UBR3 was produced using standard methods (34Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (116) Google Scholar, 38Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Smith J.A. Seidman J.G. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York2002Google Scholar, 39Harlow E. Lane D. Using Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999Google Scholar), using purified GST-UBR3-(182-598) fusion as an antigen. This antibody was employed to detect endogenous UBR3 through a combination of immunoprecipitation and immunoblotting (see Fig. 3D and “Results”). LacZ Staining, Immunohistochemistry, and Northern Hybridization—LacZ staining (using 5-bromo-4-chloro-3-in-dolyl-β-d-galactopyranoside) and Northern hybridization (with a UBR3-specific 32P-labeled cDNA probe) were carried out as described (24Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.-G. An J.Y. Seo J.W. Du F. Varshavsky A. Science. 2002; 297: 96-99Crossref PubMed Scopus (262) Google Scholar). LacZ-stained embryos were postfixed, photographed, and sectioned after embedding in paraffin. Alternatively, the tissues were frozen in OCT medium after fixation, cryosectioned, and stained for LacZ on the slides. For immunohistochemistry, specific tissues were isolated, fixed in 4% formaldehyde, treated with 70% ethanol, dehydrated, and embedded in paraffin wax as described (16Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (123) Google Scholar), followed by sectioning and staining with hematoxylin/eosin or an antibody. For Northern hybridization, a mouse adult tissue blot (Seegene, Seoul, Korea) containing total RNAs isolated from the brain, heart, lung, liver, spleen, kidney, stomach, small intestine, skeletal muscle, thymus, testis, uterus of nonpregnant female, and E17.5 placenta was hybridized with 32P-labeled cDNA fragments of mouse UBR3 or human β-actin. Northern analyses were also done with total RNAs from UBR3-/- brains and hearts versus their wild-type counterparts. Assays for Neurotransmitters, Amino Acids, and Neurotrophins—Individual brains for each UBR3 genotype (+/+, +/-, and -/-) at the stages of E18 and postnatal day 1 (P1) were collected (6-8 brains of each genotype) to measure neurotransmitters and related compounds as described (40Chourbaji S. Hellweg R. Brandis D. Zörner B. Zacher C. Lang U.E. Henn F.A. Hörtnagl H. Gass P. Brain Res. Mol. Brain Res. 2004; 121: 28-36Crossref PubMed Scopus (162) Google Scholar). The levels of monoamines (noradrenaline, dopamine, and serotonin) and their metabolites were determined using high performance liquid chromatography (HPLC) with electrochemical detection. Amino acids were measured using HPLC and fluorescence spectroscopy (41Piepponen T.P. Skujins A. J. Chromatogr. B. Biomed. Sci. Appl. 2001; 757: 277-283Crossref PubMed Scopus (80) Google Scholar). The levels of nerve growth factor and brain-derived neurotrophic factor in the brains were determined by enzyme-linked immunoassay (42Hellweg R. Baethge C. Hartung H.D. Bruckner M.K. Arendt T. Neuroreport. 1996; 7: 777-780Crossref PubMed Scopus (21) Google Scholar). Odor-based Behavioral Tests—Odorant discrimination tests were performed largely as described (43Trinh K. Storm D.R. Nat. Neurosci. 2003; 6: 519-525Crossref PubMed Scopus (188) Google Scholar) with minor modifications. UBR3-/- mice (7-17 weeks old) with littermate controls (+/+ and UBR3+/-) were individually housed in filter-topped cages 2 days before the test. On the third day, the test mouse was relocated to a designated area away from other mice and was left there for 30 min to allow accommodation to the environment. Pretraining sessions and odorant tests with mice are described under “Results.” Odorant solutions (50 μm) were made freshly in deionized water (for isoamyl acetate and ethyl vanillin) or mineral oil (for geraniol and citral). One experimenter performed the entire set of odorant tests without knowing the genotypes of mice being tested. Cloning of Mouse cDNAs Encoding UBR3—Data base search using UBR1 and UBR2 sequences as probes brought forth a 388-bp cDNA fragment (GenBank™ accession number W83893) encoding a partial open reading frame that exhibits a weak but significant homology to C-terminal regions of UBR1 and UBR2. By employing cDNA library screening and 5′-rapid amplification of cDNA ends PCR, we cloned the ∼8-kb full-length mouse cDNA, yielding nine overlapping cDNA fragments that represented two isoforms of a ∼213-kDa protein named UBR3. The predicted mouse UBR3 cDNA isoforms, encoding the proteins of 1,889 and 1,893 residues, respectively, contained distinct exon/intron boundaries, indicating alternative RNA splicing (data not shown). Reverse transcription-PCR analysis detected comparable mRNA levels o" @default.
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• W2000329656 title "Biochemical and Genetic Studies of UBR3, a Ubiquitin Ligase with a Function in Olfactory and Other Sensory Systems" @default.
• W2000329656 cites W1483715699 @default.
• W2000329656 cites W1546068374 @default.
• W2000329656 cites W1614085367 @default.
• W2000329656 cites W181940896 @default.
• W2000329656 cites W1967281537 @default.
• W2000329656 cites W1969173424 @default.
• W2000329656 cites W1973523066 @default.
• W2000329656 cites W1983345237 @default.
• W2000329656 cites W1988031203 @default.
• W2000329656 cites W1991930087 @default.
• W2000329656 cites W1992873344 @default.
• W2000329656 cites W1995430236 @default.
• W2000329656 cites W1995673848 @default.
• W2000329656 cites W1998239636 @default.
• W2000329656 cites W2010829205 @default.
• W2000329656 cites W2010909249 @default.
• W2000329656 cites W2012588962 @default.
• W2000329656 cites W2018348038 @default.
• W2000329656 cites W2021363994 @default.
• W2000329656 cites W2022387887 @default.
• W2000329656 cites W2025979901 @default.
• W2000329656 cites W2026030909 @default.
• W2000329656 cites W2027151805 @default.
• W2000329656 cites W2028819956 @default.
• W2000329656 cites W2030570400 @default.
• W2000329656 cites W2035291966 @default.
• W2000329656 cites W2038316650 @default.
• W2000329656 cites W2046290726 @default.
• W2000329656 cites W2048772732 @default.
• W2000329656 cites W2049176073 @default.
• W2000329656 cites W2052779687 @default.
• W2000329656 cites W2054182306 @default.
• W2000329656 cites W2058326770 @default.
• W2000329656 cites W2061156149 @default.
• W2000329656 cites W2061512758 @default.
• W2000329656 cites W2066168326 @default.
• W2000329656 cites W2069494008 @default.
• W2000329656 cites W2071056869 @default.
• W2000329656 cites W2077913969 @default.
• W2000329656 cites W2084587567 @default.
• W2000329656 cites W2086211835 @default.
• W2000329656 cites W2086660552 @default.
• W2000329656 cites W2093950531 @default.
• W2000329656 cites W2094572545 @default.
• W2000329656 cites W2097186487 @default.
• W2000329656 cites W2097350262 @default.
• W2000329656 cites W2098255530 @default.
• W2000329656 cites W2098933735 @default.
• W2000329656 cites W2107140412 @default.
• W2000329656 cites W2107915635 @default.
• W2000329656 cites W2108014213 @default.
• W2000329656 cites W2116880855 @default.
• W2000329656 cites W2117148991 @default.
• W2000329656 cites W2117639529 @default.
• W2000329656 cites W2128645913 @default.
• W2000329656 cites W2133617345 @default.
• W2000329656 cites W2140558722 @default.
• W2000329656 cites W2141911876 @default.
• W2000329656 cites W2166081528 @default.
• W2000329656 cites W2166802900 @default.
• W2000329656 cites W2168145001 @default.
• W2000329656 cites W4230883568 @default.
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