P19 binds to regions of short dsRNA

It is possible that P19 also binds substrates with dsRNA regions that are siRNA-like in length, but contain one strand that is significantly longer than the standard siRNA length of 21 nt. Biochemical analysis shows that RNA duplexes without 3′ overhangs bind with slightly higher affinity to P19 than their 3′-overhang-containing counterparts, indicating that the length of the RNA duplex and not the overhang is the key factor in determining P19 binding (Vargason et al. 2003). This hypothesis could explain why some molecules immunoprecipitated by P19 form no canonical siRNA-like duplexes with other cloned RNAs; short RNAs complementary to longer RNAs could be bound by P19, and the iterative size selection in the short RNA cloning protocol would exclude the longer RNA-binding partner from the final set of cloned sequences. The high-GC content of the short RNAs bound by P19 is consistent with this hypothesis (Table 1), allowing a greater promiscuity for stable binding to longer RNA partners. Indeed, in addition to enriching for short RNAs, P19 immunoprecipitation reproducibly pulls down larger RNA species that are the same size as abundant tRNAs, snRNAs, and rRNAs (Figs. 1D, ​,4A).4A). While no immunoprecipitated RNAs have exact complements in the set of known tRNAs, snRNAs, and rRNAs, gapped alignments allowing G:U pairing show that P19-enriched RNAs could form duplexes with several ncRNA species, potentially explaining the enrichment for ncRNAs as well as short rRNAs in the immunoprecipitation (not shown).

Open in a separate window

FIGURE 4.

Endogenous short rRNAs exist independently of Dicer. (A) P19 associates with short RNAs in the absence of Dicer. Dicer +/+ or −/− cells were transfected with either P19V5 or GFP as a negative control. Immunoprecipitations using V5 antibody were performed, and the associated RNA was 3′-end labeled and visualized as in Fig. 1D. (B) Similar RNA species associate with P19V5 in the presence or absence of Dicer. Shown is a short RNA Northern blot probing immunoprecipitated and supernatant RNA from A with probes complementary to RNA #3, miR295, or U6 snRNA.

We designed three different RNA duplexes to test the affinity of P19V5 for short RNAs bound to longer RNAs (Fig. 3A). One strand of each duplex was a frequently cloned short rRNA, while the other varied such that its complement was either: in the 5′ region of a 32-nt-long RNA species (5′ complementary), in the 3′ region of a 35-nt-long RNA species (3′complementary), or a 21-nt-long RNA that formed a canonical 19-bp siRNA duplex (siRNA). The length of the double-stranded region in the 5′-complementary RNA was 19 bp and was 21 bp in the 3′-complementary RNA. Since P19 preferentially binds 19-bp duplexes, the latter may be expected to have a lower affinity than the former. In all cases, the dsRNA species had 5′-phosphate and 3′-hydroxyl groups, mimicking endogenous siRNA structure. Binding assays were performed by incubating radiolabeled RNA substrate in increasing concentration with P19V5-Protein G agarose bead complexes (Fig. 3). Control experiments showed that all input RNA was double stranded and of the appropriate dilution (not shown). Under the conditions assayed, there was negligible nonspecific association of RNA with beads, and the P19V5–bead complexes had no affinity for single-stranded RNA (Fig. 3B). The apparent dissociation constant, K_app, of P19V5 for an siRNA duplex was determined to be 2.8 ± 0.5 nM, nearly identical to previously published binding studies using a P19 C-terminally tagged with GST (Fig. 3C; Lakatos et al. 2004). P19V5 bound the 5′- and 3′-complementary RNAs with K_app of 7.4 ± 0.7 nM and 27 ± 4 nM, respectively, supporting our hypothesis that P19 binds regions of dsRNA ~19 bp long and not only siRNAs (Fig. 3C).

Open in a separate window

FIGURE 3.

P19 binds with high affinity to RNAs containing 19- to 21-bp double-stranded regions with extended 5′- or 3′-single-stranded segments. (A) Base composition and secondary structure of short RNAs tested for binding to P19V5. In all cases, the strand in gray is short rRNA #3 from Fig. 2A. (B) Representative P19-binding assay. Shown is the RNA bound by P19V5–bead complexes after incubation with increasing concentrations of radiolabeled RNA (1, 5, 10, 50, and 100 nM). (C) Determination of the affinity of P19 for selected RNA species. Shown is the quantitation of a binding assay similar to that in B. The K_app was determined by fitting the average data points to a fixed endpoint curve using KaleidaGraph data analysis software.

Cell culture, transfection, and luciferase assays

J1 ES and 293T cells were grown as in Houbaviy et al. (2003) and Petersen et al. (2006). Cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For ES cell and 293T immunoprecipitation experiments, 25 μg of plasmid per 10-cm plate were used. Transfection efficiency as assessed by GFP expression was between 60% and 80% for ES cell immunoprecipitation experiments, and >95% for 293T experiments.

Dual luciferase assays were performed essentially as previously described (Doench and Sharp 2004; Petersen et al. 2006). For ES cell assays, 1 ng of each 3′-UTR reporter construct, 100 ng of pGl3 control (Promega), and 700 ng of pWhiteScript carrier DNA were transfected per well of a 24-well plate and lysed 24 h post-transfection. For 293T assays, 4 μg of plasmid (P19V5/NLS/HA or βGal control) was transfected per well of a six-well plate, and split 8 h post-transfection into a 24-well plate, seeding cells at 2 × 10⁵ cells per well. Twenty-four hours later, an additional 0.7 μg of plasmid was cotransfected along with 30 ng of pRL and pGL3 plasmids (Promega) and the appropriate amount of siRNA to attain the concentration indicated in Supplemental Figure 2. Cells were lysed 24 h post-transfection. The siRNA used perfectly targeted the coding sequence of Renilla Luciferase (target sequence: 5′-GCCAAGAAGUUUCCUAAUA-3′).

Western blots and immunohistochemistry

For western analysis, cell extracts were fractionated on 4%–20% SDS-polyacrylamide gradient gels (Biorad) and transferred to Hybond-C membranes (Amersham). Membranes were blocked with 5% milk in PBS and then incubated with the indicated antibody. Antibodies (αGAPDH [Chemicon]; αV5 [Invitrogen]; αCyclin T1 [Abcam]) were detected using HRP-conjugated antisera (Amersham) and chemiluminescence. For immunofluorescent staining, ES cells were transfected on gelatinized cover slips, then fixed with 4% PFA in PBS and permeabilized with 1% Triton X-100. Coverslips were stained with primary and secondary antibodies in PBS for 1 h and affixed to glass slides with Prolong Gold with DAPI (Molecular Probes). Actin was visualized using Alexa 488 Phalloidin (Molecular Probes).

Immunoprecipitations and RNA isolation and visualization

For each immunoprecipitation experiment, three 10-cm plates of ES cells were lysed 2 d post-transfection with WCE buffer (1% NP40, 30 mM HEPES KOH pH 7.5, 100 mM NaCl, 66 mM KCl, 1 mM MgCl₂, 1 mM DTT, 1U/μL SUPERaseIn [Ambion], Complete Protease Inhibitor EDTA-free [Roche], and Phosphatase Inhibitors I and II [Sigma]) or NE buffer (0.2% NP-40, 100 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 1U/μL SUPERaseIn, Complete Protease Inhibitor EDTA-free, and Phosphatase Inhibitors I and II). The WCE lysate was spun at 20,000 rpm in a tabletop centrifuge for 15 min after lysis to pellet insoluble material. The NE cells were lysed for 5 min at 4°C and spun for 5 min at 1000g. The pellet (containing nuclei) was resuspended in WCE buffer and sonicated on ice three times for 5 sec at power level 2 on a Branson Sonifier 450 sonicator, with a 1-min rest on ice between sonications. These sonication conditions did not disrupt endogenous short RNA binding to P19 (not shown). The sonicated nuclear fraction was spun at 20,000 rpm for 15 min in a tabletop centrifuge to remove particulate. P19 was immunoprecipitated from extracts by adding 20 μL of Protein G Plus agarose beads (Pierce) preconjugated overnight with 2 μg of V5 antibody per 10-cm plate, and rotating for 45 min at 4°C. Beads were washed with 1 mL of WCE buffer three times, switching tubes for the final wash. Beads were then resuspended in 300 μL 1× Proteinase K buffer (Nykanen et al. 2001) with 5 μL of Proteinase K (Roche) and rotated for 30 min at room temperature, then extracted with phenol/chloroform and precipitated. To isolate supernatant RNA, 300 μL of supernatant from the immunoprecipitation was extracted with phenol/chloroform and precipitated. One hundred nanograms of supernatant RNA and one-tenth the volume of immunoprecipitated RNA were 3′-end labeled with T4 RNA ligase (NEB) and 5′-α³²P 5′-3′ cytidine bis-phosphate (NEN) overnight at 4°C in 1× RNA ligase buffer and 30% v/v DMSO. Unincorporated radioactivity was removed with G25 microspin columns (Amersham), and one-half of the labeling reaction was resolved on a 12% or 15% denaturing polyacrylamide gel (National Diagnostics). For size markers, 10-bp DNA ladder (Invitrogen) or a 21-bp siRNA was 5′-end labeled using γ³²P-ATP (NEN) and T4 PNK (NEB). Gels were wrapped in Saran Wrap and quantitated on a PhosphoImager. Northern blots were performed as in Houbaviy et al. (2003), except hybridization was carried out in Oligo-Hyb (Ambion) at 37°C. Total mouse tissue RNA was purchased from Ambion. The same conditions for the ES cell WCE immunoprecipitations were used for 293T immunoprecipitations.

Binding assays

293T lysates were used to make P19-containing cell extracts for binding assays because of the lack of association between P19 and endogenous RNAs in these cells (Supplemental Fig. 2). Extracts were made 48 h post-transfection with P19V5 using 400 μL of WCE buffer per plate. Cells were lysed and extracts were spun at top speed in a tabletop centrifuge for 15 min. Protein G Plus agarose beads pre-conjugated overnight with V5 antibody were added to cleared lysate, and P19V5 was immunoprecipitated for 45 min at 4°C. Beads were washed 3× in WCE buffer and resuspended in WCE buffer such that for each binding condition, 30 μg of extract, 10 μL of 50% protein G slurry, and 0.5 μg of V5 antibody were used. Radiolabeled dsRNA was added to P19V5–bead mixtures at the indicated concentrations, and the binding reactions were rotated for 30 min at room temperature. Beads were washed 2× with 500 μL of WCE buffer, and then 2× with 1 mL of WCE buffer, switching tubes for the final wash. The bound RNA was eluted using the Proteinase K phenol/chloroform treatment described above. One-half of each reaction was resolved on a 15% polyacrylamide gel and visualized with a PhosphoImager, or quantitated via scintillation counting. Data points were then fit to a fixed-endpoint curve, (m2)*m0/(m0+m1), using KaleidaGraph software, where m2 is the maximum amount bound, m1 is the apparent dissociation constant, K_app, and m0 is the [RNA].

To make radiolabeled, duplexed RNA, 5′-phosphorylated RNA oligos (“RNA#3” 5′-CGGCUCCGGGACGGCCGGGAA-3′; “5´-complementary” 5′-CCCGGCCGUCCCGGAGCCGGCUUGGCUUCGU-3′; “3′-complementary” 5′-CUUGGCUUCGUUUCCCGGCCGUCCCGGAGCCGUU-3′; “siRNA complementary” 5′-CCCGGCCGUCCCGGAGCCGUU-3′) were annealed to their complementary strands at 12 μM in a solution containing 10 mM HEPES pH 7.5, 20 mM NaCl, and 1 mM EDTA by heating the RNAs to 95°C and cooling 1°C/min until the samples reached room temperature. Radioactive 5′-end-labeled RNA #3 was spiked into annealing reactions before heating. Dilution series of each dsRNA species were made, and portions of each were run on a 20% native polyacrylamide gel to assess efficiency of annealing and accuracy of dilution. By this analysis, all dsRNA preparations used for binding were >99% dsRNA and had an R² value for the dilution series of ≥0.99 (not shown).

We thank J. Burgyan and O. Voinnet for providing P19 plasmids, and are especially grateful to J. Neilson for protocols and help with short RNA cloning. We also thank A. Seila for help with bioinformatics, C. Whittaker for help with Figure 2, A. Leung for help with microscopy, and S. Erkeland, A. Garfinkel, J. Neilson, and A. Seila for critical reading of this manuscript. This work was supported by a Program Project Grant from the National Cancer Institute to P.A.S. and partially by the Cancer Center Support (core) grant from the National Cancer Institute.