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• W2076304893 abstract "MicroRNAs (miRNA) control numerous physiological and pathological processes. Typically, the primary miRNA (pri-miRNA) transcripts are processed by nuclear Drosha complex into ∼70-nucleotide stem-loop precursor miRNAs (pre-miRNA), which are further cleaved by cytoplasmic Dicer complex into ∼21-nucleotide mature miRNAs. However, it is unclear how nascent pre-miRNAs are protected from ribonucleases, such as MCPIP1, that degrade pre-miRNAs to abort miRNA production. Here, we identify Sjögren syndrome antigen B (SSB)/La as a pre-miRNA-binding protein that regulates miRNA processing in vitro. All three RNA-binding motifs (LAM, RRM1, and RRM2) of La/SSB are required for efficient pre-miRNA binding. Intriguingly, La/SSB recognizes the characteristic stem-loop structure of pre-miRNAs, of which the majority lack a 3′ UUU terminus. Moreover, La/SSB associates with endogenous pri-/pre-miRNAs and promotes miRNA biogenesis by stabilizing pre-miRNAs from nuclease (e.g. MCPIP1)-mediated decay in mammalian cells. Accordingly, we observed positive correlations between the expression status of La/SSB and Dicer in human cancer transcriptome and prognosis. These studies identify an important function of La/SSB as a global regulator of miRNA expression, and implicate stem-loop recognition as a major mechanism that mediates association between La/SSB and diverse RNA molecules. MicroRNAs (miRNA) control numerous physiological and pathological processes. Typically, the primary miRNA (pri-miRNA) transcripts are processed by nuclear Drosha complex into ∼70-nucleotide stem-loop precursor miRNAs (pre-miRNA), which are further cleaved by cytoplasmic Dicer complex into ∼21-nucleotide mature miRNAs. However, it is unclear how nascent pre-miRNAs are protected from ribonucleases, such as MCPIP1, that degrade pre-miRNAs to abort miRNA production. Here, we identify Sjögren syndrome antigen B (SSB)/La as a pre-miRNA-binding protein that regulates miRNA processing in vitro. All three RNA-binding motifs (LAM, RRM1, and RRM2) of La/SSB are required for efficient pre-miRNA binding. Intriguingly, La/SSB recognizes the characteristic stem-loop structure of pre-miRNAs, of which the majority lack a 3′ UUU terminus. Moreover, La/SSB associates with endogenous pri-/pre-miRNAs and promotes miRNA biogenesis by stabilizing pre-miRNAs from nuclease (e.g. MCPIP1)-mediated decay in mammalian cells. Accordingly, we observed positive correlations between the expression status of La/SSB and Dicer in human cancer transcriptome and prognosis. These studies identify an important function of La/SSB as a global regulator of miRNA expression, and implicate stem-loop recognition as a major mechanism that mediates association between La/SSB and diverse RNA molecules. MicroRNAs (miRNAs) 3The abbreviations used are: miRNAmicroRNASSBSjögren syndrome antigen BMCPIP1monocyte chemotactic protein-induced protein 1ntnucleotide(s)LAMLa motifRRMRNA recognition motifqPCRquantitative PCRGSEAgene set enrichment analysisIPimmunoprecipitateRNPribonucleoprotein. are ∼21-nucleotide (nt) cellular RNAs that govern numerous biological and disease processes by directing degradation and translational repression of cognate mRNAs (1Bartel D.P. MicroRNAs. Genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29453) Google Scholar, 2Fabian M.R. Sonenberg N. Filipowicz W. Regulation of mRNA translation and stability by microRNAs.Annu. Rev. Biochem. 2010; 79: 351-379Crossref PubMed Scopus (2304) Google Scholar, 3He L. Hannon G.J. MicroRNAs. Small RNAs with a big role in gene regulation.Nat. Rev. Genet. 2004; 5: 522-531Crossref PubMed Scopus (5624) Google Scholar). It is estimated that human genome encodes up to one thousand miRNAs (1Bartel D.P. MicroRNAs. Genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29453) Google Scholar, 3He L. Hannon G.J. MicroRNAs. Small RNAs with a big role in gene regulation.Nat. Rev. Genet. 2004; 5: 522-531Crossref PubMed Scopus (5624) Google Scholar, 4Ambros V. The functions of animal microRNAs.Nature. 2004; 431: 350-355Crossref PubMed Scopus (9048) Google Scholar), which are transcribed either as independent genes, or imbedded in other genes. The majority of miRNAs are generated by sequential processing by two RNase III enzymes: Drosha and Dicer. In the nucleus, the primary miRNA (pri-miRNA) transcripts are processed by the Drosha·DGCR8/Pasha complex into ∼70-nt stem-loop precursor miRNAs (pre-miRNAs) (5Denli A.M. Tops B.B. Plasterk R.H. Ketting R.F. Hannon G.J. Processing of primary microRNAs by the Microprocessor complex.Nature. 2004; 432: 231-235Crossref PubMed Scopus (2004) Google Scholar, 6Gregory R.I. Yan K.P. Amuthan G. Chendrimada T. Doratotaj B. Cooch N. Shiekhattar R. The Microprocessor complex mediates the genesis of microRNAs.Nature. 2004; 432: 235-240Crossref PubMed Scopus (2103) Google Scholar, 7Han J. Lee Y. Yeom K.H. Nam J.W. Heo I. Rhee J.K. Sohn S.Y. Cho Y. Zhang B.T. Kim V.N. Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex.Cell. 2006; 125: 887-901Abstract Full Text Full Text PDF PubMed Scopus (1173) Google Scholar, 8Lee Y. Ahn C. Han J. Choi H. Kim J. Yim J. Lee J. Provost P. Rådmark O. Kim S. Kim V.N. The nuclear RNase III Drosha initiates microRNA processing.Nature. 2003; 425: 415-419Crossref PubMed Scopus (3970) Google Scholar). The pre-miRNAs are exported by Exportin 5·Ran:GTP to the cytoplasm (9Lund E. Güttinger S. Calado A. Dahlberg J.E. Kutay U. Nuclear export of microRNA precursors.Science. 2004; 303: 95-98Crossref PubMed Scopus (2066) Google Scholar, 10Yi R. Qin Y. Macara I.G. Cullen B.R. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs.Genes Dev. 2003; 17: 3011-3016Crossref PubMed Scopus (2191) Google Scholar), and are further cleaved into mature miRNAs by the Dicer-1·Loqs-PB complex in Drosophila melanogaster (11Förstemann K. Tomari Y. Du T. Vagin V.V. Denli A.M. Bratu D.P. Klattenhoff C. Theurkauf W.E. Zamore P.D. Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein.PLoS Biol. 2005; 3: e236Crossref PubMed Scopus (425) Google Scholar, 12Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Dicer-1 and R3D1-L catalyze miRNA maturation in Drosophila.Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (238) Google Scholar, 13Saito K. Ishizuka A. Siomi H. Siomi M.C. Processing of pre-microRNAs by the Dicer-1-Loquacious complex in Drosophila cells.PLoS Biol. 2005; 3: e235Crossref PubMed Scopus (330) Google Scholar), or by the Dicer·TRBP complex in mammals (14Chendrimada T.P. Gregory R.I. Kumaraswamy E. Norman J. Cooch N. Nishikura K. Shiekhattar R. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing.Nature. 2005; 436: 740-744Crossref PubMed Scopus (1591) Google Scholar, 15Haase A.D. Jaskiewicz L. Zhang H. Lainé S. Sack R. Gatignol A. Filipowicz W. Mammalian Dicer finds a partner.EMBO Rep. 2005; 6: 961-967Crossref PubMed Scopus (528) Google Scholar, 16Hutvágner G. McLachlan J. Pasquinelli A.E. Bálint E. Tuschl T. Zamore P.D. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.Science. 2001; 293: 834-838Crossref PubMed Scopus (2175) Google Scholar, 17Paroo Z. Ye X. Chen S. Liu Q. Phosphorylation of the human microRNA-generating complex mediates MAPK/Erk signaling.Cell. 2009; 139: 112-122Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Both DGCR8/Pasha or TRBP/Loqs-PB are dsRNA-binding proteins that promote miRNA processing by stabilizing Drosha and Dicer and by facilitating their interaction with pri-miRNAs and pre-miRNAs, respectively (18Carthew R.W. Sontheimer E.J. Origins and Mechanisms of miRNAs and siRNAs.Cell. 2009; 136: 642-655Abstract Full Text Full Text PDF PubMed Scopus (3781) Google Scholar, 19Kim V.N. Han J. Siomi M.C. Biogenesis of small RNAs in animals.Nat. Rev. Mol. Cell Biol. 2009; 10: 126-139Crossref PubMed Scopus (2581) Google Scholar, 20Liu Q. Paroo Z. Biochemical principles of small RNA pathways.Annu. Rev. Biochem. 2010; 79: 295-319Crossref PubMed Scopus (144) Google Scholar). A number of post-transcriptional regulators of miRNA biogenesis have recently been characterized, including Lin28, KSRP, hnRNP A1, and SMAD (21Trabucchi M. Briata P. Filipowicz W. Ramos A. Gherzi R. Rosenfeld M.G. KSRP promotes the maturation of a group of miRNA precursors.Adv. Exp. Med. Biol. 2010; 700: 36-42Crossref PubMed Scopus (19) Google Scholar, 22Trabucchi M. Briata P. Garcia-Mayoral M. Haase A.D. Filipowicz W. Ramos A. Gherzi R. Rosenfeld M.G. The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs.Nature. 2009; 459: 1010-1014Crossref PubMed Scopus (518) Google Scholar, 23Davis B.N. Hilyard A.C. Lagna G. Hata A. SMAD proteins control DROSHA-mediated microRNA maturation.Nature. 2008; 454: 56-61Crossref PubMed Scopus (1120) Google Scholar, 24Michlewski G. Cáceres J.F. Antagonistic role of hnRNP A1 and KSRP in the regulation of let-7a biogenesis.Nat. Struct. Mol. Biol. 2010; 17: 1011-1018Crossref PubMed Scopus (216) Google Scholar, 25Hagan J.P. Piskounova E. Gregory R.I. Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells.Nat. Struct. Mol. Biol. 2009; 16: 1021-1025Crossref PubMed Scopus (410) Google Scholar, 26Heo I. Joo C. Cho J. Ha M. Han J. Kim V.N. Lin28 mediates the terminal uridylation of let-7 precursor MicroRNA.Mol. Cell. 2008; 32: 276-284Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar, 27Viswanathan S.R. Daley G.Q. Gregory R.I. Selective blockade of microRNA processing by Lin28.Science. 2008; 320: 97-100Crossref PubMed Scopus (1187) Google Scholar). All of these miRNA regulators modulate the production of a small subset of miRNAs through recognition of specific sequences of pri- and/or pre-miRNAs (22Trabucchi M. Briata P. Garcia-Mayoral M. Haase A.D. Filipowicz W. Ramos A. Gherzi R. Rosenfeld M.G. The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs.Nature. 2009; 459: 1010-1014Crossref PubMed Scopus (518) Google Scholar, 23Davis B.N. Hilyard A.C. Lagna G. Hata A. SMAD proteins control DROSHA-mediated microRNA maturation.Nature. 2008; 454: 56-61Crossref PubMed Scopus (1120) Google Scholar, 28Heo I. Joo C. Kim Y.K. Ha M. Yoon M.J. Cho J. Yeom K.H. Han J. Kim V.N. TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation.Cell. 2009; 138: 696-708Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar). microRNA Sjögren syndrome antigen B monocyte chemotactic protein-induced protein 1 nucleotide(s) La motif RNA recognition motif quantitative PCR gene set enrichment analysis immunoprecipitate ribonucleoprotein. In many RNA maturation processes, the precursor molecules are often bound by RNA-binding proteins or chaperones to protect them from nuclease-mediated decay and to ensure the correct processing by specific ribonucleases. In the current model, however, newly synthesized pri-/pre-miRNAs are described as unprotected and delivered directly to Drosha or Dicer enzymes for processing. It has recently been suggested that the processing of pri-miRNAs by Drosha occurs co-transcriptionally on chromatin (29Morlando M. Ballarino M. Gromak N. Pagano F. Bozzoni I. Proudfoot N.J. Primary microRNA transcripts are processed co-transcriptionally.Nat. Struct. Mol. Biol. 2008; 15: 902-909Crossref PubMed Scopus (297) Google Scholar, 30Pawlicki J.M. Steitz J.A. Primary microRNA transcript retention at sites of transcription leads to enhanced microRNA production.J. Cell Biol. 2008; 182: 61-76Crossref PubMed Scopus (115) Google Scholar). In contrast, nascent pre-miRNAs embark a long and perilous journey from the nucleus to the cytoplasm, in which they are vulnerable to degradation by many nucleases. For example, the monocyte chemotactic protein-induced protein 1 (MCPIP1)/ZC3H12A, a ribonuclease and deubiquitinase that suppresses inflammatory response (31Matsushita K. Takeuchi O. Standley D.M. Kumagai Y. Kawagoe T. Miyake T. Satoh T. Kato H. Tsujimura T. Nakamura H. Akira S. Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA decay.Nature. 2009; 458: 1185-1190Crossref PubMed Scopus (476) Google Scholar, 32Liang J. Saad Y. Lei T. Wang J. Qi D. Yang Q. Kolattukudy P.E. Fu M. MCP-induced protein 1 deubiquitinates TRAF proteins and negatively regulates JNK and NF-κB signaling.J. Exp. Med. 2010; 207: 2959-2973Crossref PubMed Scopus (231) Google Scholar), was recently reported to antagonize Dicer by degrading pre-miRNAs to abort miRNA production (33Suzuki H.I. Arase M. Matsuyama H. Choi Y.L. Ueno T. Mano H. Sugimoto K. Miyazono K. MCPIP1 ribonuclease antagonizes dicer and terminates microRNA biogenesis through precursor microRNA degradation.Mol. Cell. 2011; 44: 424-436Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Sjögren syndrome antigen B (SSB), also known as autoantigen La, is commonly associated with autoimmune disorders such as Sjögren syndrome and systemic lupus erythematosus (34Mattioli M. Reichlin M. Heterogeneity of RNA protein antigens reactive with sera of patients with systemic lupus erythematosus. Description of a cytoplasmic nonribosomal antigen.Arthritis Rheum. 1974; 17: 421-429Crossref PubMed Scopus (214) Google Scholar). The La/SSB protein is evolutionarily conserved from yeast to human, exists abundantly in both the nucleus and cytoplasm, and plays fundamental roles in diverse processes of RNA metabolism (35Wolin S.L. Cedervall T. The La protein.Annu. Rev. Biochem. 2002; 71: 375-403Crossref PubMed Scopus (337) Google Scholar). A well known function of La/SSB is its association with nascent transcripts of RNA polymerase III, including precursors of tRNAs (pre-tRNAs), through recognition of the characteristic 3′ UUU termini. The binding of La/SSB protects pre-tRNAs from nonspecific exonuclease digestion and ensures correct processing of the 5′ and 3′ leader sequences of pre-tRNAs by specific ribonucleases, such as RNase P and RNase Z (36Chakshusmathi G. Kim S.D. Rubinson D.A. Wolin S.L. A La protein requirement for efficient pre-tRNA folding.EMBO J. 2003; 22: 6562-6572Crossref PubMed Scopus (96) Google Scholar, 37Fan H. Goodier J.L. Chamberlain J.R. Engelke D.R. Maraia R.J. 5′ processing of tRNA precursors can be modulated by the human La antigen phosphoprotein.Mol. Cell. Biol. 1998; 18: 3201-3211Crossref PubMed Scopus (101) Google Scholar, 38Yoo C.J. Wolin S.L. The La protein in Schizosaccharomyces pombe. A conserved yet dispensable phosphoprotein that functions in tRNA maturation.Cell. 1997; 89: 393-402Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Both Drosophila and human La/SSB contain three RNA-binding motifs, a highly conserved La motif (LAM) (39Bousquet-Antonelli C. Deragon J.M. A comprehensive analysis of the La-motif protein superfamily.RNA. 2009; 15: 750-764Crossref PubMed Scopus (133) Google Scholar), a canonical RNA recognition motif (RRM1), and an atypical RRM2, as well as a variable carboxyl (C) terminus. It has previously been shown that the amino (N)-terminal LAM-RRM1 domain of La/SSB is responsible for 3′ UUU recognition (40Alfano C. Sanfelice D. Babon J. Kelly G. Jacks A. Curry S. Conte M.R. Structural analysis of cooperative RNA binding by the La motif and central RRM domain of human La protein.Nat. Struct. Mol. Biol. 2004; 11: 323-329Crossref PubMed Scopus (111) Google Scholar, 41Kotik-Kogan O. Valentine E.R. Sanfelice D. Conte M.R. Curry S. Structural analysis reveals conformational plasticity in the recognition of RNA 3′ ends by the human La protein.Structure. 2008; 16: 852-862Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 42Teplova M. Yuan Y.R. Phan A.T. Malinina L. Ilin S. Teplov A. Patel D.J. Structural basis for recognition and sequestration of UUU(OH) 3′ temini of nascent RNA polymerase III transcripts by La, a rheumatic disease autoantigen.Mol. Cell. 2006; 21: 75-85Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Although La/SSB is dispensable for yeast viability (43Copela L.A. Chakshusmathi G. Sherrer R.L. Wolin S.L. The La protein functions redundantly with tRNA modification enzymes to ensure tRNA structural stability.RNA. 2006; 12: 644-654Crossref PubMed Scopus (51) Google Scholar, 44Yoo C.J. Wolin S.L. La proteins from Drosophila melanogaster and Saccharomyces cerevisiae. A yeast homolog of the La autoantigen is dispensable for growth.Mol. Cell. Biol. 1994; 14: 5412-5424Crossref PubMed Scopus (106) Google Scholar), it is essential in higher eukaryotes such as Drosophila and mice (45Park J.M. Kohn M.J. Bruinsma M.W. Vech C. Intine R.V. Fuhrmann S. Grinberg A. Mukherjee I. Love P.E. Ko M.S. DePamphilis M.L. Maraia R.J. The multifunctional RNA-binding protein La is required for mouse development and for the establishment of embryonic stem cells.Mol. Cell. Biol. 2006; 26: 1445-1451Crossref PubMed Scopus (46) Google Scholar, 46Bai C. Tolias P.P. Genetic analysis of a La homolog in Drosophila melanogaster.Nucleic Acids Res. 2000; 28: 1078-1084Crossref PubMed Scopus (20) Google Scholar). The molecular basis for this discrepancy is currently unclear. Here, we have uncovered an important function for La/SSB in promoting global miRNA expression by binding and stabilizing pre-miRNAs. Moreover, we showed that La/SSB interacts with pre-miRNAs by stem-loop recognition rather than 3′ UUU recognition. These significant findings provide fresh insights into the basic process of miRNA biogenesis, the diverse functions of La/SSB in RNA metabolism, and possibly the pathogenesis of cancer and autoimmune diseases. General RNA reagents were purchased from Ambion and Promega, and DNA oligos or siRNAs were synthesized by IDT. Both Dicer and human La/SSB antibodies were purchased from Santa Cruz Biotechnology, whereas actin, tubulin, and anti-FLAG antibodies were obtained from Sigma. Recombinant Drosophila Dicer-1·Loqs-PB and human Dicer·TRBP complexes were generated in insect cells as previously described (12Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Dicer-1 and R3D1-L catalyze miRNA maturation in Drosophila.Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (238) Google Scholar, 17Paroo Z. Ye X. Chen S. Liu Q. Phosphorylation of the human microRNA-generating complex mediates MAPK/Erk signaling.Cell. 2009; 139: 112-122Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Recombinant Drosophila and human La/SSB were produced in Escherichia coli and purified by nickel affinity and ion-exchange chromatography. Recombinant FLAG-tagged MCPIP1 and Drosha·DGCR8 complex was affinity purified from 293T cells following transient transfection (47Han J. Lee Y. Yeom K.H. Kim Y.K. Jin H. Kim V.N. The Drosha-DGCR8 complex in primary microRNA processing.Genes Dev. 2004; 18: 3016-3027Crossref PubMed Scopus (1581) Google Scholar). The cytoplasmic extract (S100) of Drosophila S2 cells (48Liu Y. Ye X. Jiang F. Liang C. Chen D. Peng J. Kinch L.N. Grishin N.V. Liu Q. C3PO, an endoribonuclease that promotes RNAi by facilitating RISC activation.Science. 2009; 325: 750-753Crossref PubMed Scopus (188) Google Scholar) was precipitated by ammonium sulfate at 60% saturation. After a 30-min, 20,000 × g centrifugation step, the supernatant was dialyzed overnight in Buffer A (10 mm KOAc, 10 mm HEPES, pH 7.4, 2 mm Mg(OAc)2, 2.5 mm DTT), loaded onto a Mono S column, and eluted with a 300–600 mm NaCl gradient. The fractions with peak activity were dialyzed for 4 h, loaded onto a Mono Q column, and eluted with a 0–300 mm NaCl gradient. Finally, the peak fractions were directly loaded on a Smart Mono S column and eluted with a 300–600 mm NaCl gradient. HeLa cell extract was prepared in RIPA buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 1 mm EDTA) freshly supplemented with protease inhibitors. HeLa extract was incubated with anti-human La/SSB monoclonal antibody or mouse IgG for 1 h at 4 °C, and protein G-Sepharose beads were added and rotated for another 4 h. After extensive wash of the beads (4 times in RIPA buffer, 3 times in RIPA buffer with 0.5 m NaCl, and once with buffer III (0.25 m LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mm EDTA, 10 mm Tris-HCl, pH 8.0), associated RNA was extracted from the beads by TRIzol and precipitated by ethanol. To demonstrate in vivo association between La and pre-miRNAs, we transfected HeLa cells with a empty vector or FLAG-hLa/SSB expression construct. Thirty-six hours after transfection, HeLa cells were either untreated or treated with 1.0% formaldehyde before making protein extracts in RIPA buffer (49Niranjanakumari S. Lasda E. Brazas R. Garcia-Blanco M.A. Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo.Methods. 2002; 26: 182-190Crossref PubMed Scopus (334) Google Scholar). The FLAG-La RNP complexes were immunoprecipitated from lysates using anti-FLAG (M2) antibody-conjugated affinity gel (Sigma). After extensive washing in a highly stringent condition (RIPA buffer containing 1 m NaCl and 4 m urea), the beads were resuspended in reversal buffer (50 mm Tris-Cl, pH 7.0, 5 mm EDTA, 10 mm DTT, and 1% SDS) and cooked at 70 °C for 1 h. Associated RNA was extracted by TRIzol (Invitrogen), and followed by RT-PCR to detect endogenous pre-miRNAs. The PCR primers for amplifying pri/pre-miRNAs were listed in Table 1.TABLE 1siRNA targets, primer sequences, and Northern probesPrimer sequencessiLa1 (human) target sequence5′-GGUCGUAGAUUUAAAGGAA-3′siLa2 (human) target sequence5′-GGUUAGAAGAUAAAGGUCA-3′Cyclophillin (human) forward5′-TGCCATCGCCAAGGAGTAG-3′Cyclophillin (human) reverse5′-TGCACAGACGGTCACTCAAA-3′pri-let-7a (human) forward5′-CTCATTACACAGGAAACCGGAA-3′pri-let-7a (human) reverse5′-CCTCATCCCACAGTGAAGAGAA-3′pri-miR-16 (human) forward5′-CTGACATGCTTGTTCCACTCTAGC-3′pri-miR-16 (human) reverse5′-CCTGTCACACTAAAGCAGCACAAT-3′pri-miR-17 (human) forward5′-GTTGTTAGAGTTTGAGGTGTT-3′pri-miR-17 (human) reverse5′-AGCACTCAACATCAGCAGG-3′pri-miR-21 (human) forward5′-TACCATCGTGACATCTCCA-3′pri-miR-21 (human) reverse5′-CAGACAGAAGGACCAGAGTT-3′pri-miR-23b (human) forward5′-CAGTGTGTGCAGACAGCAC-3′pri-miR-23b (human) reverse5′-GTTCTCCAATCTGCAGTGA-3′pre-let-7a (human) forward5′-TGGGATGAGGTAGTAGGTTGT-3′pre-let-7a (human) reverse5′-TAGGAAAGACAGTAGATTGTATAGTT-3′pre-miR-21 (human) forward5′-TAGCTTATCAGACTGATGTTGA-3′pre-miR-21 (human) reverse5′-CGACTGCTGTTGCCATGAG-3′pre-miR-23b (human) forward5′-CAGGTGCTCTGGCTGCTT-3′pre-miR-23b (human) reverse5′-GTGGTAATCCCTGGCAATGT-3′Anti-5.8S (human)5′-TCCTGCAATTCACATTAATTCTCGCAG-3′Anti-5S (human)5′-CCGACCCTGCTTAGCTTCCGAGATCA-3′Anti-U6S (human)5′-CGTTCCAATTTTAGTATATGTGCTGCC-3′Anti-miR-165′-CGCCAAUAUUUACGUGCUGCUA-3′Anti-miR-175′-CUACCUGCACUGUAAGCACUUUG-3′Anti-miR-205′-CUACCUGCACUAUAAGCACUUUA-3′Anti-miR-215′-UCAACAUCAGUCUGAUAAGCUA-3′Anti-miR-23b5′-GGUAAUCCCUGGCAAUGUGAU-3′Anti-miR-305′-CUUCCAGUCGAGGAUGUUUACU-3′Anti-let-7a5′-AACUAUACAACCUACUACCUCA-3′ Open table in a new tab For siRNA-mediated knockdown in HeLa cells, 1 × 106 of cells were plated in a 10-cm dish and transfected with different siRNAs (siGFP, siLa1, or siLa2) by Lipofectamine RNAi Max (Invitrogen). After 2 days, transfected cells were replated at 1 × 106 cells/10-cm dish followed by a second siRNA transfection. Three days later, cells were harvested and total RNA was extracted by TRIzol. To generate the Teton-inducible La knockdown cells, we stably integrated a transgene that encodes four copies of a small hairpin RNA (shLa, same target sequence as siLa 2, Table 1) under control of an inducible H1 promoter, into U2OS cells expressing Tet repressor (Oligoengine). Doxycycline was added to the medium (2 μm final concentration) to induce the expression of small hairpin RNA (shLa). The U2OS/shLa (FLAG-dLa) rescue cell line was constructed by stable integration of a FLAG-tagged Drosophila La cDNA transgene into the U2OS/shLa cell line. Both HeLa and U2OS cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 100 μg/ml of streptomycin. Northern blotting for pre-miRNA and miRNA detection was performed essentially as described (50Liu X. Park J.K. Jiang F. Liu Y. McKearin D. Liu Q. Dicer-1, but not Loquacious, is critical for assembly of miRNA-induced silencing complexes.RNA. 2007; 13: 2324-2329Crossref PubMed Scopus (50) Google Scholar). Typically, 30 μg of total RNA was resolved by 12% urea-PAGE and transferred to GT membrane (Bio-Rad) followed by UV cross-linking. All miRNA probes are 21-nt ssDNA or RNA oligos (IDT) that are complementary to miRNA sequences provided by miRBase. The 5S, 5.8S, and U6 probe sequences are listed in Table 1. All probes were 5′ radiolabeled with [γ-32P]ATP by T4 polynucleotide kinase (New England Biolabs). Hybridization was conducted in ultrasensitive hybridization buffer (Ambion) at 40 °C overnight. The membrane was washed 3 times at 40 °C in 2× SSC, 0.5% SDS, and exposed to x-ray film. For RT-PCR, cDNAs were generated using the reverse transcription kit (Applied Biosystems) and amplified by PCR with Taq polymerase. The primer sequences are listed in Table 1. TaqMan qPCR was performed to measure the levels of specific miRNAs or pri-miRNAs according to the manufacturer's protocol (Applied Biosystems). Relative quantities were calculated using the ΔΔCt method. 18S rRNA served as a loading control. Global miRNA profiling was performed using MegaPlex RT Primers and TaqMan Array microRNA Cards (Applied Biosystems, Human Pool A version 2.1 MegaPlex RT primers and Human MicroRNA A Cards version 2.0) according to the manufacturer's protocols. 750 ng of total RNA was used in Megaplex RT without pre-amplification. U6 snRNA served as the endogenous control as the average Ct varied by ≤0.2 between control and experimental treatments. Threshold was held constant at 0.2 ΔRn and baseline was set from cycles 3 to 16 for all miRNA. miRNA with average Ct ≥35 or abnormal amplification curves in control and experimental treatments were omitted and considered as not expressed. Average relative quantities were calculated among replicates and a t test was used to identify miRNA differentially expressed based on a p value threshold of p ≤ 0.05. Synthetic pre-miRNAs were 5′ radiolabeled with [γ-32P]ATP by T4 polynucleotide kinase followed by G-25 column (Ambion) purification. The pri-let-7a-1 construct, a gift from Dr. Narry Kim, was used to generate uniformly radiolabeled pri-miRNA substrate by in vitro transcription followed by PAGE purification. Typically, recombinant proteins and radiolabeled RNA were incubated at 37 (human proteins) or 30 °C (Drosophila proteins) for 30 min in a 10-μl reaction (100 mm KOAc, 15 mm HEPES, pH 7.4, 2.5 mm EDTA, 2.5 mm DTT, pH 7.4). The reaction mixture was resolved by a 5% native PAGE and exposed to x-ray film. Gene expression profiling data of breast cancer patients were obtained from the NCBI Gene Expression Omnibus (GSE7390) and bioinformatics.nki.nl/data.php. The expression levels of Dicer, La/SSB, or MCPIP1 were evaluated by the corresponding probes. Genes negatively or positively associated with Dicer expression (“Dicer high/low down-regulated or up-regulated genes”) were defined as 200 of the most differentially expressed genes between the top 30 cases with high and low Dicer expression. After dividing all cases into halves according to La expression, GSEA was performed with GSEA software available from the Broad Institute (51Subramanian A. Tamayo P. Mootha V.K. Mukherjee S. Ebert B.L. Gillette M.A. Paulovich A. Pomeroy S.L. Golub T.R. Lander E.S. Mesirov J.P. Gene set enrichment analysis. A knowledge-based approach for interpreting genome-wide expression profiles.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15545-15550Crossref PubMed Scopus (26549) Google Scholar). Survival analysis was performed using the survival package of R, the survfit function, and the survdiff function. Classification of high or low expression of Dicer, La/SSB, and MCPIP1 was performed as previously described (52Martello G. Rosato A. Ferrari F. Manfrin A. Cordenonsi M. Dupont S. Enzo E. Guzzardo V. Rondina M. Spruce T. Parenti A.R. Daidone M.G. Bicciato S. Piccolo S. A MicroRNA targeting Dicer for metastasis control.Cell. 2010; 141: 1195-1207Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar). We took an unbiased biochemical approach to identify new regulators of miRNA biogenesis by supplementing recombinant Dicer-1·Loqs-PB complex (Fig. 1A) with the fractions of cytoplasmic extract (S100) of Drosophila S2 cells. We observed an inhibitory activity of pre-miRNA processing in the supernatant after 60% saturation of ammonium sulfate precipitation of S2/S100 (Fig. 1B). This factor was further purified to homogeneity by sequential chromatographic fractionation (Fig. 1C). At the final purification step, only a single protein of ∼50 kDa appeared on the silver-stained polyacrylamide gel (PAGE), and correlated closely with the miRNA regulatory activity (Fig. 1C). This protein was identified as the Drosophila homolog of La/SSB by mass spectrometric analysis. We generated His-tagged Drosophila (dLa) and human (hLa) La/SSB recombinant proteins (Fig. 1D). Addition of dLa efficiently suppressed the ability of the recombinant Dicer-1·Loqs-PB complex to process pre-miRNA into miRNA in vitro (Fig. 1E). Likewise, hLa could inhibit pre-miRNA processing by the recombinant Dicer·TRBP complex (Fig. 1F). Intriguingly, we obtained a strikingly different result when performing pre-miRNA processing assays using crude cell extracts. Addition of La/SSB could instead stabilize pre-miRNA and enhance miRNA production in both S2 and H" @default.
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• W2076304893 date "2013-01-01" @default.
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• W2076304893 title "Sjögren Syndrome Antigen B (SSB)/La Promotes Global MicroRNA Expression by Binding MicroRNA Precursors through Stem-Loop Recognition" @default.
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