Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M705208200 on July 31, 2007

J. Biol. Chem., Vol. 282, Issue 39, 28373-28378, September 28, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/39/28373    most recent
M705208200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ye, X.
Right arrow Articles by Liu, Q.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ye, X.
Right arrow Articles by Liu, Q.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Functional Anatomy of the Drosophila MicroRNA-generating Enzyme*

Xuecheng Ye, Zain Paroo, and Qinghua Liu, A W. A. "Tex" Moncrief, Jr. Scholar in Medical Research and a Damon Runyon Scholar, supported by the Damon Runyon Cancer Research Foundation (Grant DRS-43)1

From the Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9038

Received for publication, June 25, 2007 , and in revised form, July 30, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
RNA interference is a post-transcriptional gene silencing mechanism mediated by microRNAs (miRNAs)2 and small interfering RNAs (siRNAs) (1–5). In the initiation step, siRNAs and miRNAs are generated respectively from mostly exogenous long double-stranded RNA (dsRNA) and endogenous short hairpin pre-miRNA (6, 7). In the effector step, nascent siRNA and miRNA are assembled into similar RNA-induced silencing complexes termed siRISC and miRISC (8, 9). 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 (6, 10, 11).

Biogenesis of miRNAs involves two RNase III enzymes: Drosha and Dicer (12). In the nucleus, the primary transcript (pri-miRNA) of a miRNA gene is processed by Drosha into ~60-nucleotide stem-loop pre-miRNA (13). Drosha requires the assistance of a dsRNA-binding protein, known as Pasha or DGCR8, to accurately process pri-miRNA to pre-miRNA (14–17). The pre-miRNA is then exported by Exportin 5 to the cytoplasm (18–20), where it is further cleaved by Dicer into miRNA (7, 21). 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 (22). 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 (23).

In Drosophila melanogaster, two Dicer enzymes, Dcr-1 and Dcr-2, are responsible for miRNA and siRNA production, respectively (8, 21, 24). Despite extensive sequence homology, Dcr-1 and Dcr-2 display distinct substrate specificities and ATP requirements (8). 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 (8). 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 (5). For example, Dcr-2 and R2D2 constitute the Drosophila, siRNA-generating enzyme (24). R2D2 contains two dsRBD domains and forms a heterodimeric complex with Dcr-2 (24). Although R2D2 does not regulate siRNA production, it cooperates with Dcr-2 to promote assembly of the effector siRISC complexes (24–26). Only the Dcr-2-R2D2 complex, neither Dcr-2 nor R2D2 alone, could efficiently interact with duplex siRNA (25). Both Dcr-2 and R2D2 are critical components of the RISC loading complex, the formation of which precedes and is required for RISC activation (27–29).

Loqacious (Loqs) functions as a co-factor for Dcr-1 in the Drosophila miRNA pathway (8, 30, 31). 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 (30). Both Loqs-PA and Loqs-PB carry three dsRBD domains (8, 30). 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 (8). 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 (8). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (8). 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 (8, 24). 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 2x 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 [{gamma}-32P] ATP (Valeant Pharmaceuticals) or at the 3' end by T4 RNA ligase (Ambion) and [{alpha}-32P] pCp (Amersham Biosciences) followed by PAGE purification. Typically, 5 x 104 cpm pre-miRNA was incubated with recombinant Dcr-1 enzymes at 30 °C for 30 min in 10-µl reactions (25). For the kinetic studies (see Fig. 3g), 2.5 x 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 x 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 (8).

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 4x 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (22). 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 (8).

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).


Figure 1
View larger version (50K):
[in this window]
[in a new window]

  		FIGURE 1.
Dcr-1 contains one processing center. a, sequence alignment between the two RIII domains of human Dicer and Drosophila Dcr-1. The catalytic residues are boxed. b, top, an SDS-PAGE gel showing purified wild type (WT) and five catalytic mutant Dcr-1 recombinant proteins. The pre-miRNA-processing assays were conducted by incubating recombinant Dcr-1 proteins with a 5'-radiolabeled (middle) or 3'-radiolabeled (bottom) pre-let-7 substrate. DM, double mutant. c, a schematic model of pre-miRNA processing by wild type (top), RIIIa mutants (middle), or RIIIb mutants (bottom) Dcr-1.

 


Figure 2
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 2.
Dcr-1 exists as a monomer. Western blots were performed to detect Dcr-1 in individual fractions following sedimentation analysis of recombinant Dcr-1 (top), Dcr-1-Loqs-PB complex (middle), or cytoplasmic (S100) extract of S2 cells (bottom) on a 10–30% glycerol gradient. The positions of molecular mass markers are listed above: {gamma}-globulin (158 kDa) and Ferritin (440 kDa).

 
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 (8, 30). 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 (8). 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.


Figure 3
View larger version (53K):
[in this window]
[in a new window]

  		FIGURE 3.
Processing of pre-miRNA by truncated Dcr-1. a, a schematic diagram of full-length (FL) and truncated (T1–T6) Dcr-1. The numbers in parentheses refer to amino acid positions. b, Coomassie Brilliant Blue-stained SDS-PAGE gel showing purified full-length (lane 1) and truncated (lanes 2–6) His-Dcr-1 recombinant proteins. c and d, the pre-miRNA-processing assays were performed by incubating 0.2 pmol of recombinant Dcr-1 and 5'-radiolabeled pre-let7 (c) or pre-bantam (d) substrate. Reaction time was 30 min. e, Coomassie Brilliant Blue-stained SDS-PAGE showing purified FL and T6 recombinant Dcr-1. f, the pre-miRNA-processing assays were performed using 0.02, 0.04, 0.1, or 0.2 pmol of FL and T6 recombinant Dcr-1. Reaction time was 30 min. g, kinetic studies of pre-miRNA processing to compare the activities of (0.2 pmol) FL, T1, T5, and T6 of recombinant Dcr-1 enzymes. The numbers in parentheses correspond to the relative percentage of wild type Dcr-1 activity.

 


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 4.
Dcr-1 preferentially associates with Loqs-PB. a, insect cells were infected with His-tagged Loqs-PB (lanes 1–2), Loqs-PA (lanes 3–4), or Loqs-PA/Loqs-PB (lanes 5–6) viruses in the absence (odd lanes) or presence (even lanes) of the FLAG-Dcr-1 virus. IP was conducted using anti-FLAG antibodies. Western blots were performed with anti-FLAG (top) or anti-His (middle and bottom) antibodies. b, Co-IP experiments were performed with S2 extracts using mouse monoclonal anti-Dcr-1, anti-His, or anti-Loqs antibodies followed by Western blots with rabbit polyclonal anti-Loqs antibodies.

 
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 (8). 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.


Figure 5
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 5.
Mapping of the interaction domains between Dcr-1 and Loqs. a, insect cells were infected with His-tagged FL or truncated (T1–T5) Dcr-1 viruses (Fig. 3a) in the absence (odd lanes) or presence (even lanes) of the FLAG-Loqs-PB virus. Immunoprecipitation (IP) was conducted with anti-FLAG antibodies followed by Western blots using anti-His antibodies. A small amount of His-Dcr-1 proteins non-specifically associated with the anti-FLAG beads. b, a schematic diagram of FL and truncated (L1–L5) Loqs proteins. The numbers in parentheses correspond to amino acid positions. c, S2 cells were transfected with constructs expressing Myc-tagged helicase domain of Dcr-1 and GFP-FL or L1–L4 of Loqs-PB. d, S2 cells were transfected with constructs expressing full-length Myc-Dcr-1 and GFP-L4 (lane 1), GFP-L5 (lane 2), or GFP-L4/GFP-L5 (lane 3). Western blots were performed with anti-GFP or anti-Myc antibodies.

 


Figure 6
View larger version (40K):
[in this window]
[in a new window]

  		FIGURE 6.
Loqs-PB requires the second and third dsRBDs to regulate the activity of Dcr-1. a, Coomassie Brilliant Blue-stained SDS-PAGE showing recombinant FL and truncated (L1–L4) Loqs-PB proteins. b, the pre-miRNA processing assays were conducted using wild type Dcr-1 alone or with FL or truncated (L1–L4) Loqs-PB. c, the pre-miRNA gel-shift assays were performed by incubating radiolabeled pre-miRNA with the catalytic mutant (E1908A/D2139) Dcr-1 or in combination with full-length or truncated (L1–L4) Loqs-PB. d, the photocross-linking assays were performed by incubating radiolabeled pre-miRNA with Dcr-1 (E1908A/D2139) in the presence of full-length, truncated (L3 and L4), or dsRBD2 mutant (L3m) Loqs-PB followed by exposure to UV light. The reaction mixtures were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and exposed to x-ray film. e, Western blots were performed with anti-His antibody using the same membrane in d. f, the pre-miRNA-processing assays were performed with recombinant Dcr-1 alone or in the presence of L3 or L3m.

 
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 (8). 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 (24). 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.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The primitive Giardia Dicer carries only the PAZ domain and two RIII domains but displays robust dsRNA-processing activity (23). 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 (32). 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 (33). Thus, the Loqs-PB, but not Loqs-PA, isoform is necessary and sufficient for Drosophila development and the miRNA pathway (33). 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 (8, 24).

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 (8). 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 (34) 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.


   FOOTNOTES
 
* This work was supported in part by a Welch grant (I-1608) and National Institute of Health grants awarded to Q. L. (GM078163). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd. K3.219, Dallas, TX 75390-9038. Tel.: 214-648-9120; Fax: 214-648-8856; E-mail: Qinghua.Liu{at}UTsouthwestern.edu.

2 The abbreviations used are: miRNA, microRNAs; siRNA, small interfering RNA; dsRNA, double-stranded RNA; dsRBD, dsRNA-binding domain; pri, primary; pre, precursor; Loqs, Loquacious; Dcr-1, Dicer-1; RIII, RNase III; RISC, RNA-induced silencing complex; GFP, green fluorescent protein; IP, immunoprecipitation; Co-IP, co-immunoprecipitation; FL, full-length; T, truncated Dcr-1; L, truncated Loqs. Back


   ACKNOWLEDGMENTS
 
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.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hannon, G. J. (2002) Nature 418, 244-251[CrossRef][Medline] [Order article via Infotrieve]
  2. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Nature 391, 806-811[CrossRef][Medline] [Order article via Infotrieve]
  3. Tomari, Y., and Zamore, P. D. (2005) Genes Dev. 19, 517-529[Abstract/Free Full Text]
  4. Lee, R. C., Feinbaum, R. L., and Ambros, V. (1993) Cell 75, 843-854[CrossRef][Medline] [Order article via Infotrieve]
  5. Paroo, Z., Liu, Q., and Wang, X. (2007) Cell Res. 17, 187-194[Medline] [Order article via Infotrieve]
  6. Zamore, P. D., Tuschl, T., Sharp, P. A., and Bartel, D. P. (2000) Cell 101, 25-33[CrossRef][Medline] [Order article via Infotrieve]
  7. Hutvagner, G., McLachlan, J., Pasquinelli, A. E., Balint, E., Tuschl, T., and Zamore, P. D. (2001) Science 293, 834-838[Abstract/Free Full Text]
  8. Jiang, F., Ye, X., Liu, X., Fincher, L., McKearin, D., and Liu, Q. (2005) Genes Dev. 19, 1674-1679[Abstract/Free Full Text]
  9. Tang, G. (2005) Trends Biochem. Sci. 30, 106-114[CrossRef][Medline] [Order article via Infotrieve]
  10. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Nature 411, 494-498[CrossRef][Medline] [Order article via Infotrieve]
  11. Hutvagner, G., and Zamore, P. D. (2002) Science 297, 2056-2060[Abstract/Free Full Text]
  12. Cullen, B. R. (2004) Mol. Cell 16, 861-865[CrossRef][Medline] [Order article via Infotrieve]
  13. Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P., Radmark, O., Kim, S., and Kim, V. N. (2003) Nature 425, 415-419[CrossRef][Medline] [Order article via Infotrieve]
  14. Gregory, R. I., Yan, K. P., Amuthan, G., Chendrimada, T., Doratotaj, B., Cooch, N., and Shiekhattar, R. (2004) Nature 432, 235-240[CrossRef][Medline] [Order article via Infotrieve]
  15. Han, J., Lee, Y., Yeom, K. H., Kim, Y. K., Jin, H., and Kim, V. N. (2004) Genes Dev. 18, 3016-3027[Abstract/Free Full Text]
  16. Landthaler, M., Yalcin, A., and Tuschl, T. (2004) Curr. Biol. 14, 2162-2167[CrossRef][Medline] [Order article via Infotrieve]
  17. Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F., and Hannon, G. J. (2004) Nature 432(7014), 231-235[CrossRef][Medline] [Order article via Infotrieve]
  18. Bohnsack, M. T., Czaplinski, K., and Gorlich, D. (2004) RNA (Cold Spring Harbor) 10, 185-191
  19. Yi, R., Qin, Y., Macara, I. G., and Cullen, B. R. (2003) Genes Dev. 17, 3011-3016[Abstract/Free Full Text]
  20. Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E., and Kutay, U. (2004) Science 303(5654), 95-98[Abstract/Free Full Text]
  21. Lee, Y. S., Nakahara, K., Pham, J. W., Kim, K., He, Z., Sontheimer, E. J., and Carthew, R. W. (2004) Cell 117, 69-81[CrossRef][Medline] [Order article via Infotrieve]
  22. Zhang, H., Kolb, F. A., Jaskiewicz, L., Westhof, E., and Filipowicz, W. (2004) Cell 118, 57-68[CrossRef][Medline] [Order article via Infotrieve]
  23. Macrae, I. J., Zhou, K., Li, F., Repic, A., Brooks, A. N., Cande, W. Z., Adams, P. D., and Doudna, J. A. (2006) Science 311(5758), 195-198[Abstract/Free Full Text]
  24. Liu, Q., Rand, T. A., Kalidas, S., Du, F., Kim, H. E., Smith, D. P., and Wang, X. (2003) Science 301(5641), 1921-1925[Abstract/Free Full Text]
  25. Liu, X., Jiang, F., Kalidas, S., Smith, D., and Liu, Q. (2006) RNA (Cold Spring Harbor) 12, 1514-1520
  26. Tomari, Y., Matranga, C., Haley, B., Martinez, N., and Zamore, P. D. (2004) Science 306(5700), 1377-1380[Abstract/Free Full Text]
  27. Tomari, Y., Du, T., Haley, B., Schwarz, D. S., Bennett, R., Cook, H. A., Koppetsch, B. S., Theurkauf, W. E., and Zamore, P. D. (2004) Cell 116, 831-841[CrossRef][Medline] [Order article via Infotrieve]
  28. Pham, J. W., Pellino, J. L., Lee, Y. S., Carthew, R. W., and Sontheimer, E. J. (2004) Cell 117, 83-94[CrossRef][Medline] [Order article via Infotrieve]
  29. Pham, J. W., and Sontheimer, E. J. (2005) J. Biol. Chem. 280, 39278-39283[Abstract/Free Full Text]
  30. Forstemann, K., Tomari, Y., Du, T., Vagin, V. V., Denli, A. M., Bratu, D. P., Klattenhoff, C., Theurkauf, W. E., and Zamore, P. D. (2005) PLoS Biol. 3, e236[CrossRef][Medline] [Order article via Infotrieve]
  31. Saito, K., Ishizuka, A., Siomi, H., and Siomi, M. C. (2005) PLoS Biol. 3, e235[CrossRef][Medline] [Order article via Infotrieve]
  32. Lee, Y., Hur, I., Park, S. Y., Kim, Y. K., Suh, M. R., and Kim, V. N. (2006) EMBO J. 25, 522-532[CrossRef][Medline] [Order article via Infotrieve]
  33. Park, J. K., Liu, X., Strauss, T. J., McKearin, D. M., and Liu, Q. (2007) Curr. Biol. 17, 533-538[CrossRef][Medline] [Order article via Infotrieve]
  34. Dlakic, M. (2006) Bioinformatics (Oxf.) 22, 2711-2714[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/39/28373    most recent
M705208200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ye, X.
Right arrow Articles by Liu, Q.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ye, X.
Right arrow Articles by Liu, Q.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement