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• W2080442032 abstract "In Drosophila melanogaster, the multidomain RNase III Dicer-1 (Dcr-1) functions in tandem with the double-stranded (ds)RNA-binding protein Loquacious (Loqs) to catalyze the maturation of microRNAs (miRNAs) from precursor (pre)-miRNAs. Here we dissect the molecular mechanism of pre-miRNA processing by the Dcr-1-Loqs complex. The tandem RNase III (RIII) domains of Dcr-1 form an intramolecular dimer such that one RIII domain cleaves the 3′ strand, whereas the other cuts the 5′ strand of pre-miRNA. We show that the functional core of Dcr-1 consists of a DUF283 domain, a PAZ domain, and two RIII domains. Dcr-1 preferentially associates with the Loqs-PB splice isoform. Loqs-PB uses the second dsRNA-binding domain to bind pre-miRNA and the third dsRNA-binding domain to interact with Dcr-1. Both domains of Loqs-PB are required for efficient miRNA production by enhancing the affinity of Dcr-1 for pre-miRNA. Thus, our results provide further insights into the functional anatomy of the Drosophila miRNA-generating enzyme. In Drosophila melanogaster, the multidomain RNase III Dicer-1 (Dcr-1) functions in tandem with the double-stranded (ds)RNA-binding protein Loquacious (Loqs) to catalyze the maturation of microRNAs (miRNAs) from precursor (pre)-miRNAs. Here we dissect the molecular mechanism of pre-miRNA processing by the Dcr-1-Loqs complex. The tandem RNase III (RIII) domains of Dcr-1 form an intramolecular dimer such that one RIII domain cleaves the 3′ strand, whereas the other cuts the 5′ strand of pre-miRNA. We show that the functional core of Dcr-1 consists of a DUF283 domain, a PAZ domain, and two RIII domains. Dcr-1 preferentially associates with the Loqs-PB splice isoform. Loqs-PB uses the second dsRNA-binding domain to bind pre-miRNA and the third dsRNA-binding domain to interact with Dcr-1. Both domains of Loqs-PB are required for efficient miRNA production by enhancing the affinity of Dcr-1 for pre-miRNA. Thus, our results provide further insights into the functional anatomy of the Drosophila miRNA-generating enzyme. RNA interference is a post-transcriptional gene silencing mechanism mediated by microRNAs (miRNAs) 2The abbreviations used are: miRNAmicroRNAssiRNAsmall interfering RNAdsRNAdouble-stranded RNAdsRBDdsRNA-binding domainpriprimarypreprecursorLoqsLoquaciousDcr-1Dicer-1RIIIRNase IIIRISCRNA-induced silencing complexGFPgreen fluorescent proteinIPimmunoprecipitationCo-IPco-immunoprecipitationFLfull-lengthTtruncated Dcr-1Ltruncated Loqs. and small interfering RNAs (siRNAs) (1Hannon G.J. Nature. 2002; 418: 244-251Crossref PubMed Scopus (3544) Google Scholar, 2Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11908) Google Scholar, 3Tomari Y. Zamore P.D. Genes Dev. 2005; 19: 517-529Crossref PubMed Scopus (750) Google Scholar, 4Lee R.C. Feinbaum R.L. Ambros V. Cell. 1993; 75: 843-854Abstract Full Text PDF PubMed Scopus (9830) Google Scholar, 5Paroo Z. Liu Q. Wang X. Cell Res. 2007; 17: 187-194Crossref PubMed Scopus (39) Google Scholar). In the initiation step, siRNAs and miRNAs are generated respectively from mostly exogenous long double-stranded RNA (dsRNA) and endogenous short hairpin pre-miRNA (6Zamore P.D. Tuschl T. Sharp P.A. Bartel D.P. Cell. 2000; 101: 25-33Abstract Full Text Full Text PDF PubMed Google Scholar, 7Hutvagner G. McLachlan J. Pasquinelli A.E. Balint E. Tuschl T. Zamore P.D. Science. 2001; 293: 834-838Crossref PubMed Scopus (2190) Google Scholar). In the effector step, nascent siRNA and miRNA are assembled into similar RNA-induced silencing complexes termed siRISC and miRISC (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar, 9Tang G. Trends Biochem. Sci. 2005; 30: 106-114Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). In RISCs, a single-stranded siRNA or miRNA functions as the guide RNA to direct sequence-specific degradation and/or translational repression of cognate mRNA (6Zamore P.D. Tuschl T. Sharp P.A. Bartel D.P. Cell. 2000; 101: 25-33Abstract Full Text Full Text PDF PubMed Google Scholar, 10Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8186) Google Scholar, 11Hutvagner G. Zamore P.D. Science. 2002; 297: 2056-2060Crossref PubMed Scopus (1649) Google Scholar). microRNAs small interfering RNA double-stranded RNA dsRNA-binding domain primary precursor Loquacious Dicer-1 RNase III RNA-induced silencing complex green fluorescent protein immunoprecipitation co-immunoprecipitation full-length truncated Dcr-1 truncated Loqs. Biogenesis of miRNAs involves two RNase III enzymes: Drosha and Dicer (12Cullen B.R. Mol. Cell. 2004; 16: 861-865Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar). In the nucleus, the primary transcript (pri-miRNA) of a miRNA gene is processed by Drosha into ∼60-nucleotide stem-loop pre-miRNA (13Lee Y. Ahn C. Han J. Choi H. Kim J. Yim J. Lee J. Provost P. Radmark O. Kim S. Kim V.N. Nature. 2003; 425: 415-419Crossref PubMed Scopus (4026) Google Scholar). Drosha requires the assistance of a dsRNA-binding protein, known as Pasha or DGCR8, to accurately process pri-miRNA to pre-miRNA (14Gregory R.I. Yan K.P. Amuthan G. Chendrimada T. Doratotaj B. Cooch N. Shiekhattar R. Nature. 2004; 432: 235-240Crossref PubMed Scopus (2130) Google Scholar, 15Han J. Lee Y. Yeom K.H. Kim Y.K. Jin H. Kim V.N. Genes Dev. 2004; 18: 3016-3027Crossref PubMed Scopus (1615) Google Scholar, 16Landthaler M. Yalcin A. Tuschl T. Curr. Biol. 2004; 14: 2162-2167Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, 17Denli A.M. Tops B.B. Plasterk R.H. Ketting R.F. Hannon G.J. Nature. 2004; 432: 231-235Crossref PubMed Scopus (2039) Google Scholar). The pre-miRNA is then exported by Exportin 5 to the cytoplasm (18Bohnsack M.T. Czaplinski K. Gorlich D. RNA (Cold Spring Harbor). 2004; 10: 185-191Google Scholar, 19Yi R. Qin Y. Macara I.G. Cullen B.R. Genes Dev. 2003; 17: 3011-3016Crossref PubMed Scopus (2222) Google Scholar, 20Lund E. Guttinger S. Calado A. Dahlberg J.E. Kutay U. Science. 2004; 303: 95-98Crossref PubMed Scopus (2089) Google Scholar), where it is further cleaved by Dicer into miRNA (7Hutvagner G. McLachlan J. Pasquinelli A.E. Balint E. Tuschl T. Zamore P.D. Science. 2001; 293: 834-838Crossref PubMed Scopus (2190) Google Scholar, 21Lee Y.S. Nakahara K. Pham J.W. Kim K. He Z. Sontheimer E.J. Carthew R.W. Cell. 2004; 117: 69-81Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar). Drosha cleaves at the base of pre-miRNA to excise it from pri-miRNA. Dicer cuts again near the loop of pre-miRNA to produce mature miRNA. Dicer is a conserved family of ∼200-kDa multidomain RNase III (RIII) enzymes. The canonical prokaryotic RNase III contains a single RIII domain and functions as a homodimer. In contrast, a typical eukaryotic Dicer consists of a DExH helicase domain, a domain of unknown function (DUF)283, and a PAZ domain at the N terminus as well as two RIII domains and a dsRNA-binding domain (dsRBD) at the C terminus. Recent biochemical studies have shown that human Dicer operates as a monomer and that its tandem RIII domains form one processing center and cleave the opposite strand of dsRNA (22Zhang H. Kolb F.A. Jaskiewicz L. Westhof E. Filipowicz W. Cell. 2004; 118: 57-68Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar). This model is supported by the x-ray crystal structure of Dicer (Protein Data Base number 2FFL) from the human parasite Giardia intestinalis. The primitive Giardia Dicer only carries a PAZ domain followed by tandem RIII domains. As Dicer resembles the shape of a hatchet, the two RIII domains form the blade as an intramolecular dimer that is similar to the homodimeric structure of prokaryotic RNase III (23Macrae I.J. Zhou K. Li F. Repic A. Brooks A.N. Cande W.Z. Adams P.D. Doudna J.A. Science. 2006; 311: 195-198Crossref PubMed Scopus (701) Google Scholar). In Drosophila melanogaster, two Dicer enzymes, Dcr-1 and Dcr-2, are responsible for miRNA and siRNA production, respectively (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar, 21Lee Y.S. Nakahara K. Pham J.W. Kim K. He Z. Sontheimer E.J. Carthew R.W. Cell. 2004; 117: 69-81Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar, 24Liu Q. Rand T.A. Kalidas S. Du F. Kim H.E. Smith D.P. Wang X. Science. 2003; 301: 1921-1925Crossref PubMed Scopus (562) Google Scholar). Despite extensive sequence homology, Dcr-1 and Dcr-2 display distinct substrate specificities and ATP requirements (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar). Dcr-1 prefers to process pre-miRNA to miRNA in an ATP-independent manner. Dcr-2 is much better at processing dsRNA and requires ATP hydrolysis for efficient siRNA production (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar). By contrast, human and most other organisms contain a single Dicer that generates both miRNAs and siRNAs. Typically, Dicer functions in tandem with a specific dsRNA-binding protein (5Paroo Z. Liu Q. Wang X. Cell Res. 2007; 17: 187-194Crossref PubMed Scopus (39) Google Scholar). For example, Dcr-2 and R2D2 constitute the Drosophila, siRNA-generating enzyme (24Liu Q. Rand T.A. Kalidas S. Du F. Kim H.E. Smith D.P. Wang X. Science. 2003; 301: 1921-1925Crossref PubMed Scopus (562) Google Scholar). R2D2 contains two dsRBD domains and forms a heterodimeric complex with Dcr-2 (24Liu Q. Rand T.A. Kalidas S. Du F. Kim H.E. Smith D.P. Wang X. Science. 2003; 301: 1921-1925Crossref PubMed Scopus (562) Google Scholar). Although R2D2 does not regulate siRNA production, it cooperates with Dcr-2 to promote assembly of the effector siRISC complexes (24Liu Q. Rand T.A. Kalidas S. Du F. Kim H.E. Smith D.P. Wang X. Science. 2003; 301: 1921-1925Crossref PubMed Scopus (562) Google Scholar, 25Liu X. Jiang F. Kalidas S. Smith D. Liu Q. RNA (Cold Spring Harbor). 2006; 12: 1514-1520Google Scholar, 26Tomari Y. Matranga C. Haley B. Martinez N. Zamore P.D. Science. 2004; 306: 1377-1380Crossref PubMed Scopus (470) Google Scholar). Only the Dcr-2-R2D2 complex, neither Dcr-2 nor R2D2 alone, could efficiently interact with duplex siRNA (25Liu X. Jiang F. Kalidas S. Smith D. Liu Q. RNA (Cold Spring Harbor). 2006; 12: 1514-1520Google Scholar). Both Dcr-2 and R2D2 are critical components of the RISC loading complex, the formation of which precedes and is required for RISC activation (27Tomari Y. Du T. Haley B. Schwarz D.S. Bennett R. Cook H.A. Koppetsch B.S. Theurkauf W.E. Zamore P.D. Cell. 2004; 116: 831-841Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 28Pham J.W. Pellino J.L. Lee Y.S. Carthew R.W. Sontheimer E.J. Cell. 2004; 117: 83-94Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 29Pham J.W. Sontheimer E.J. J. Biol. Chem. 2005; 280: 39278-39283Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Loqacious (Loqs) functions as a co-factor for Dcr-1 in the Drosophila miRNA pathway (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar, 30Forstemann K. Tomari Y. Du T. Vagin V.V. Denli A.M. Bratu D.P. Klattenhoff C. Theurkauf W.E. Zamore P.D. PLoS Biol. 2005; 3: e236Crossref PubMed Scopus (426) Google Scholar, 31Saito K. Ishizuka A. Siomi H. Siomi M.C. PLoS Biol. 2005; 3: e235Crossref PubMed Scopus (333) Google Scholar). The loqs gene produces alternative spliced transcripts encoding three protein isoforms (PA-PC). All three isoforms are expressed in S2 cells, whereas only PA and PB are expressed in fly tissues (30Forstemann K. Tomari Y. Du T. Vagin V.V. Denli A.M. Bratu D.P. Klattenhoff C. Theurkauf W.E. Zamore P.D. PLoS Biol. 2005; 3: e236Crossref PubMed Scopus (426) Google Scholar). Both Loqs-PA and Loqs-PB carry three dsRBD domains (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar, 30Forstemann K. Tomari Y. Du T. Vagin V.V. Denli A.M. Bratu D.P. Klattenhoff C. Theurkauf W.E. Zamore P.D. PLoS Biol. 2005; 3: e236Crossref PubMed Scopus (426) Google Scholar). Loqs-PB is slightly larger than Loqs-PA by 46 amino acids. As shown by biochemical fractionation of S2 extracts, Dcr-1 and Loqs-PB, but not Loqs-PA or Loqs-PC, correlate perfectly with the miRNA-generating activity (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar). Although recombinant Dcr-1 is able to process pre-miRNA to miRNA, Loqs-PB greatly enhances miRNA production by increasing the affinity of Dcr-1 for pre-miRNA (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar). Similar to Loqs, TRBP and PACT contain three dsRBD domains and have recently been identified as two dsRNA-binding proteins for human Dicer. In the current study, we performed detailed domain structure and functional analyses to dissect the molecular mechanism of pre-miRNA processing by the Drosophila miRNA-generating (Dcr-1-Loqs) enzyme. Cloning and Expression of Recombinant Protein—All truncated constructs were generated by PCR and subcloning using full-length Dcr-1, Loqs-PA, and Loqs-PB cDNA as templates (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar). The catalytic mutants of Dcr-1 were constructed by using “QuikChange” (Stratagene). Recombinant His- or FLAG-tagged full-length or truncated Dcr-1-Loqs proteins were produced in insect cells using the BAC-to-BAC baculovirus expression system (Invitrogen). Large scale productions of recombinant Dcr-1 proteins were conducted as described previously (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar, 24Liu Q. Rand T.A. Kalidas S. Du F. Kim H.E. Smith D.P. Wang X. Science. 2003; 301: 1921-1925Crossref PubMed Scopus (562) Google Scholar). For interaction studies in insect cells, 200 μl of each virus was added to 10 ml of 106 cells/ml Sf21 cells in a T25 flask. Cell extracts were prepared in the lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100) 40 h after viral infection. For interaction studies in S2 cells, expression constructs for full-length or truncated Myc-Dcr-1 and GFP-Loqs were generated by the “Gateway” system (Invitrogen). Transient transfections of S2 cells were conducted using Cellfectin (Invitrogen). Cell lysates were prepared 40 h after transfection. Antibodies and Immunoprecipitation (IP)—Mouse monoclonal anti-Dcr-1 and anti-Loqs antibodies were generated against purified recombinant Dcr-1-Loqs-PB complex. The polyclonal anti-GFP antibodies were purchased from Invitrogen, whereas monoclonal anti-FLAG (M2), anti-Myc (9E10), and anti-His (HIS-1) antibodies were from Sigma. For co-IPs, ∼2 mg of cell extract was incubated with 5 μl of antibodies at 4 °C for 2 h followed by the addition of 10 μl pre-washed protein A beads (Santa Cruz Biotechnology). After rotating at 4 °C overnight, the beads were washed six times with the IP wash buffer (110 mm potassium acetate, 10 mm HEPES, pH 7.4, 100 mm NaCl, 2 mm Mg(OAc)2, 5 mm dithiothreitol, and 1% Nonidet P-40) and boiled in 2× SDS sample buffer for 5 min. Sedimentation Analysis—In parallel, 300 μl (10 μg/μl) of S100 extract of S2 cells and 50 nm purified recombinant Dcr-1 or Dcr-1-Loqs-PB complex were applied onto a 3-ml 10–30% glycerol gradient. The contents were centrifuged at 55,000 rpm at 4 °C for 3.5 h using an SW60Ti rotor (Beckman). Fourteen fractions (250 μl/fraction) were taken from the top of the tube and subsequently used for Western blotting to detect the presence of Dcr-1. The Pre-miRNA Processing Assay—Synthetic pre-let-7 and pre-bantam RNA (Dharmacon) were radiolabeled at the 5′ end by T4 polynucleotide kinase (New England Biochemicals) and [γ-32P] ATP (Valeant Pharmaceuticals) or at the 3′ end by T4 RNA ligase (Ambion) and [α-32P] pCp (Amersham Biosciences) followed by PAGE purification. Typically, 5 × 104 cpm pre-miRNA was incubated with recombinant Dcr-1 enzymes at 30 °C for 30 min in 10-μl reactions (25Liu X. Jiang F. Kalidas S. Smith D. Liu Q. RNA (Cold Spring Harbor). 2006; 12: 1514-1520Google Scholar). For the kinetic studies (see Fig. 3g), 2.5 × 105 cpm (∼0.2 pmol) pre-miRNA was incubated with 0.2 pmol of recombinant Dcr-1 enzymes. The Pre-miRNA Gel-shift Assay—Recombinant Dcr-1-Loqs-PB proteins were incubated with 5 × 105 cpm of 5′-radiolabeled pre-miRNA at 30 °C for 30 min in the same buffer as the processing assay. The reaction mixtures were resolved on a 6% native PAGE as described previously (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar). The UV Cross-linking Assay—The 20-μl pre-miRNA gelshift reactions were performed as described above followed by exposure to UV light for 20 min in a 96-well dish. After adding 5 μl of 4× SDS sample buffer, the mixture was boiled for 5 min, resolved on a 4–20% SDS-PAGE, and transferred to cellulose membrane followed by autoradiography. Two RNase III Domains of Dcr-1 Form an Intramolecular Dimer—Previous studies have revealed that the two RIII domains of human Dicer form an intramolecular dimer and cleave the opposite strands of dsRNA (22Zhang H. Kolb F.A. Jaskiewicz L. Westhof E. Filipowicz W. Cell. 2004; 118: 57-68Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar). To determine whether Drosophila Dcr-1 had a similar action mechanism, we generated a series of catalytic mutant Dcr-1 enzymes. Based on the sequence alignment, Asp-1749 and Glu-1908 of RIIIa and Asp-2036 and Glu-2139 of RIIIb were predicted to be the corresponding catalytic residues of Dcr-1 (Fig. 1a). We inactivated either or both of the RIII domains of Dcr-1 by changing these residues to alanine by site-directed mutagenesis. Wild type and mutant His-tagged Dcr-1 recombinant proteins were produced using an insect cell expression system and highly purified by Ni2+ affinity column followed by Q-Sepharose and SP-Sepharose chromatography (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar). We compared the cleavage patterns of wild type and various catalytic mutant Dcr-1 enzymes by in vitro pre-miRNA-processing assays using either a 5′-radiolabeled or a 3′-radiolabeled substrate. Mutations of both RIII domains (E1908A/E2139) completely abolished the ability of Dcr-1 to process pre-miRNA (Fig. 1b, compare lane 1 and lane 6). Interestingly, the RIIIa (D1749A or E1908A) mutants could only cleave the 5′ (top) strand, but not the 3′ (bottom) strand, of pre-miRNA (Fig. 1b, compare lane 1 and lanes 2 and 3). Conversely, the RIIIb (D2036A or E2139A) mutants were able to cut the 3′ strand, but not the 5′ strand, of pre-miRNA (Fig. 1b, compare lane 1 and lanes 4 and 5). The mirroring cleavage patterns indicate that 5′-RIIIa cleaves the 3′ strand, whereas 3′-RIIIb cuts the 5′ strand of pre-miRNA. Thus, like human Dicer, the tandem RIII domains of Dcr-1 form one processing center. The staggered pair of cuts made by RIIIa and RIIIb excises miRNA from pre-miRNA and creates a characteristic two-nucleotide 3′ overhang terminus (Fig. 1c). Dcr-1 Exists as a Monomer—We characterized the sizes of the endogenous and recombinant Dcr-1 proteins by sedimentation analysis. On a 10–30% glycerol gradient, the Dcr-1 protein in S2 extracts fractionated with a peak of ∼400 kDa (Fig. 2). The data suggested that the bulk of endogenous Dcr-1 existed as a monomeric form and in complex with other cellular components, such as Loqs and Ago1 (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar, 30Forstemann K. Tomari Y. Du T. Vagin V.V. Denli A.M. Bratu D.P. Klattenhoff C. Theurkauf W.E. Zamore P.D. PLoS Biol. 2005; 3: e236Crossref PubMed Scopus (426) Google Scholar). At an equivalent concentration, recombinant Dcr-1 and Dcr-1-Loqs-PB complex migrated at positions that were consistent with a Dcr-1 monomer and a Dcr-1-Loqs-PB heterodimer (Fig. 2). Together, these results indicate that Dcr-1 functions as a monomer at physiological condition. Pre-miRNA Processing by Truncated Dcr-1—Dcr-1 is a multidomain RNase III enzyme that contains a helicase domain, a DUF283 domain, a PAZ domain, two RIII domains, and a dsRBD domain. To determine which of these domains were critical for Dcr-1 function, we generated a series of truncated (T1–T6) Dcr-1 and compared their activities to that of wild type enzyme by pre-miRNA-processing assays (Fig. 3, a and b). As shown in Fig. 3, c and d, removal of the DUF283 domain (T2) and/or the PAZ domain (T3) completely abolished the ability of Dcr-1 to generate miRNAs. Similar results were obtained whether using a pre-let-7 (miRNA located at the 5′ strand) (Fig. 3c) or pre-bantam (miRNA at the 3′ strand) substrate (Fig. 3d). In contrast, Dcr-1 could efficiently process pre-miRNA to miRNA without the N-terminal helicase domain (T1) or the C-terminal dsRBD domain (T5). Intriguingly, deletion of both domains (T6) still resulted in a functional Dcr-1 enzyme (Fig. 3, e and f). Further kinetic studies revealed that T1, T5, and T6 retained ∼38, 61, or 15% of the miRNA-generating activity of a wild type Dcr-1 enzyme (Fig. 3g). Dcr-1 Preferentially Associates with Loqs-PB—As shown by coimmunoprecipitation (co-IP) experiments, FLAG-tagged Dcr-1 could form a complex with either His-tagged Loqs-PA or His-tagged Loqs-PB in insect cells following co-infection of baculoviruses (Fig. 4a). Intriguingly, when insect cells were co-infected with all three viruses, Dcr-1 associated Loqs-PB almost exclusively, although Loqs-PA and Loqs-PB were expressed at equivalent levels (Fig. 4a). This result suggests that the two Loqs isoforms compete for Dcr-1 binding and that Dcr-1 may have higher affinity for Loqs-PB than Loqs-PA. We further examined the association between the endogenous Dcr-1 and Loqs proteins by co-IPs using anti-Dcr-1 and anti-Loqs monoclonal antibodies. Although anti-Loqs antibodies brought down all three Loqs (PA, PB, PC) proteins from S2 extracts, Loqs-PB was the predominant isoform detected in the IPs of anti-Dcr-1 antibodies (Fig. 4b). This was consistent with our previous observation that Dcr-1 and Loqs-PB, but not Loqs-PA or Loqs-PC, correlated perfectly with the miRNA-generating activity (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar). Together, these biochemical results indicate that Dcr-1 and Loqs-PB constitute the miRNA-generating enzyme. Interaction Domains between Dcr-1 and Loqs—To determine how Dcr-1 interacted with Loqs, we examined the interaction between FLAG-Loqs-PB and various His-tagged truncated Dcr-1 proteins in insect cells (Fig. 3a). Following co-infection of the Dcr-1-Loqs baculoviruses, IPs were performed with anti-FLAG antibodies followed by Western blotting with anti-His antibodies. As shown in Fig. 5a, Loqs-PB was unable to interact with T1, T2, or T3 of Dcr-1, all of which lacked the N-terminal helicase domain. Conversely, Loqs-PB interacted strongly with the full-length, T4, or T5 of Dcr-1 that shared the helicase domain in common. These results indicate that Loqs interacts with the helicase domain of Dcr-1. Next, we generated four truncated Loqs-PB constructs to map the region of Loqs that interacted with Dcr-1 (Fig. 5b). We performed co-IP experiments to examine the interaction between Myc-tagged helicase domain (Myc-helciase) of Dcr-1 and GFP-tagged truncated Loqs (L1–L4) in S2 cells after co-transfection of the Dcr-1-Loqs expression constructs. The anti-GFP antibodies specifically brought down Myc-helicase only in the presence of L3 (dsRBD2-dsRBD3) or L4 (dsRBD3), but not L1 (dsRBD1-dsRBD2) or L2 (dsRBD2) (Fig. 5c). Therefore, the Dcr-1-Loqs-PB association involves the N-terminal helicase domain of Dcr-1 and the C-terminal dsRBD (dsRBD3) of Loqs. Loqs-PB is larger than Loqs-PA by 46 amino acids that immediately precede the C-terminal dsRBD domain. To determine whether these 46 residues were responsible for high affinity binding between Loqs-PB and Dcr-1, we generated the L5 construct by specifically removing the 46 residues from L4. As shown in Fig. 5d, full-length Myc-Dcr-1 could interact individually with either GFP-L4 or GFP-L5, which represented the minimal Dcr-1 interaction domains of Loqs-PB and Loqs-PA, respectively. However, Myc-Dcr-1 preferentially associated with GFP-L4 rather than GFP-L5 when all three constructs were co-transfected into S2 cells. Thus, the miniature L4 and L5 constructs recapitulate the difference in Dcr-1 affinity between Loqs-PB and Loqs-PA. Loqs-PB Requires Its Second and Third dsRBDs for Dcr-1 Regulation—Although Dcr-1 alone is able to process pre-miRNA to miRNA, Loqs-PB greatly enhances miRNA production by increasing the affinity of Dcr-1 for pre-miRNA (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar). To determine which portions of Loqs-PB were critical for Dcr-1 regulation, we made recombinant full-length and various truncated Loqs-PB proteins (Fig. 6a) and compared their abilities to enhance Dcr-1 activity in the pre-miRNA-processing assay (Fig. 6b). As expected, L1 and L2 did not affect miRNA production because they lacked the third dsRBD domain that interacted with Dcr-1. Although L4 (dsRBD3) interacted with Dcr-1, it was unable to increase miRNA production. Only L3 (dsRBD2-dsRBD3) retained the ability to enhance the miRNA-generating activity of Dcr-1. These results indicate that the second and third dsRBDs of Loqs-PB are necessary and sufficient for Dcr-1 regulation. In parallel, native gel-shift assays were conducted to compare the pre-miRNA affinity of recombinant Dcr-1 in the absence or presence of full-length and truncated Loqs-PB (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar). In these assays, we employed the catalytic mutant (E1908A/E2139) Dcr-1 to prevent the cleavage of radiolabeled pre-miRNA. As shown in Fig. 6c, neither L1 nor L2 could increase the binding of Dcr-1 to pre-miRNA. On the other hand, L4 modestly increased, whereas L3 greatly enhanced the binding of Dcr-1 to pre-miRNA. We performed photocross-linking experiments to examine the physical interaction between pre-miRNA and recombinant Dcr-1-Loqs-PB proteins. As shown in Fig. 6, d and e, both Dcr-1 and Loqs-PB were efficiently cross-linked to radiolabeled pre-miRNA after exposure to ultraviolet light. In presence of Dcr-1, only L3 (dsRBD2-dsRBD3), but not L4 (dsRBD3), could be cross-linked to pre-miRNA. Furthermore, we specifically inactivated the dsRBD2 of Loqs in L3 by mutating two conserved alanines (Ala-308, Ala-309) to lysine residues as described previously for R2D2 (24Liu Q. Rand T.A. Kalidas S. Du F. Kim H.E. Smith D.P. Wang X. Science. 2003; 301: 1921-1925Crossref PubMed Scopus (562) Google Scholar). These point mutations abolished the ability of L3 to either interact with pre-miRNA or enhance miRNA production (Fig. 6, d–f). Together, these results indicate that Loqs-PB uses its dsRBD2 to bind pre-miRNA and dsRBD3 to interact with Dcr-1. Both domains of Loqs-PB are required for its ability to regulate Dcr-1 activity. The primitive Giardia Dicer carries only the PAZ domain and two RIII domains but displays robust dsRNA-processing activity (23Macrae I.J. Zhou K. Li F. Repic A. Brooks A.N. Cande W.Z. Adams P.D. Doudna J.A. Science. 2006; 311: 195-198Crossref PubMed Scopus (701) Google Scholar). Here we show that Drosophila Dcr-1 can efficiently process pre-miRNA to miRNA without the N-terminal helicase and the C-terminal dsRBD domains. By contrast, removal of the PAZ and/or the DUF283 domains completely abolished the ability of Dcr-1 to generate miRNA. A similar phenomenon has been observed for human Dicer (32Lee Y. Hur I. Park S.Y. Kim Y.K. Suh M.R. Kim V.N. EMBO J. 2006; 25: 522-532Crossref PubMed Scopus (529) Google Scholar). Thus, we propose that the functional core of eukaryotic Dicer consists of a DUF283 domain, a PAZ domain, and two RIII domains. Both Loqs-PA and Loqs-PB use the third dsRBD domain to interact with the helicase domain of Dcr-1. However, Dcr-1 preferentially associates with Loqs-PB rather than Loqs-PA when both isoforms are co-expressed. As shown by biochemical fractionation, Dcr-1 and Loqs-PB, but not Loqs-PA, correlate perfectly with the miRNA-generating activity. Furthermore, transgenic expression of Loqs-PB, but not Loqs-PA, can rescue both developmental and miRNA-processing defects of homozygous loqs knock-out flies (33Park J.K. Liu X. Strauss T.J. McKearin D.M. Liu Q. Curr. Biol. 2007; 17: 533-538Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Thus, the Loqs-PB, but not Loqs-PA, isoform is necessary and sufficient for Drosophila development and the miRNA pathway (33Park J.K. Liu X. Strauss T.J. McKearin D.M. Liu Q. Curr. Biol. 2007; 17: 533-538Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Taken together, our previous and current studies demonstrate that the Dcr-1-Loqs-PB and Dcr-2-R2D2 complexes catalyze Drosophila miRNA and siRNA biogenesis, respectively (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar, 24Liu Q. Rand T.A. Kalidas S. Du F. Kim H.E. Smith D.P. Wang X. Science. 2003; 301: 1921-1925Crossref PubMed Scopus (562) Google Scholar). Although R2D2 does not regulate the siRNA production of Dcr-2, Loqs-PB greatly enhances miRNA production by increasing the affinity of Dcr-1 for pre-miRNA (8Jiang F. Ye X. Liu X. Fincher L. McKearin D. Liu Q. Genes Dev. 2005; 19: 1674-1679Crossref PubMed Scopus (240) Google Scholar). The current study suggests a simple model of pre-miRNA processing by the Dcr-1-Loqs complex. On its own, Dcr-1 is able to generate miRNA. The minimal requirements for Dcr-1 to process pre-miRNA include a DUF283 domain and a central PAZ domain followed by two RIII domains. The PAZ domain is believed to interact with the terminus of pre-miRNA stem. The DUF283 domain is a dsRNA-binding domain (34Dlakic M. Bioinformatics (Oxf.). 2006; 22: 2711-2714Crossref PubMed Scopus (51) Google Scholar) and may be critical for the binding or processing of pre-miRNA. The tandem RIII domains form an intramolecular dimer such that RIIIa cleaves the 3′ strand, whereas RIIIb cleaves the 5′ strand of pre-miRNA. The pair of cuts creates a two-nucleotide 3′ overhang and excises miRNA from pre-miRNA. On the other hand, Loqs-PB uses the C-terminal dsRBD3 to interact with the N-terminal helicase domain of Dcr-1. The middle dsRBD2 of Loqs-PB is responsible for direct binding to pre-miRNA. Interestingly, although L4 (dsRBD3) of Loqs-PB does not physically contact pre-miRNA, it modestly enhances the binding of Dcr-1 to pre-miRNA. One possible explanation is that the binding of dsRBD3 to Dcr-1 triggers a conformational change in Dcr-1, thereby increasing its affinity for pre-miRNA. These specific interactions greatly enhance the miRNA-generating activity of Dcr-1, possibly by strengthening the interaction between Dcr-1 and pre-miRNA. Additionally, they may organize the enzyme/substrate in a proper conformation for efficient processing. Our biochemical studies provide further insights into the functional anatomy of the Drosophila miRNA-generating enzyme. The results may help understand the process of miRNA biogenesis in other model organisms. We thank Dr. Wayne Lai at the antibody core facility for generating monoclonal antibodies and Drs. Yi Liu and Gaya Amarasinghe for discussion and critical comments on the manuscript." @default.
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