Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs

Abstract

microRNAs (miRNAs) regulate the expression of mRNAs in animals and plants through miRNA-containing ribonucleoprotein particles (RNPs). At the core of these miRNA silencing effector complexes are the Argonaute (AGO) proteins that bind miRNAs and mediate target mRNA recognition. We generated HEK293 cell lines stably expressing epitope-tagged human AGO proteins and other RNA silencing-related proteins and used these cells to purify miRNA-containing RNPs. Mass spectrometric analyses of the proteins associated with different AGO proteins revealed a common set of helicases and mRNA-binding proteins, among them the three trinucleotide repeat containing proteins 6 (TNRC6A,-B,-C). mRNA microarray analyses of these miRNA-associated RNPs revealed that AGO and TNRC6 proteins bind highly similar sets of transcripts enriched in binding sites for highly expressed endogenous miRNAs, indicating that the TNRC6 proteins are a component of the mRNA-targeting miRNA silencing complex. Together with the very similar proteomic composition of each AGO complex, this result suggests substantial functional redundancy within families of human AGO and TNRC6 proteins. Our results further demonstrate that we have developed an effective biochemical approach to identify physiologically relevant human miRNA targets.

FIGURE 1.

Proteins identified by mass spectrometry interact with EIF2C1. HEK293 cells stably expressing FLAG/HA-EIF2C1 were transfected with constructs encoding myc-tagged variants of the proteins identified by MS. Transfected cells were lysed and cytoplasmic extracts prepared in the absence or presence (+RNase T1) of ribonuclease T1. FLAG/HA-EIF2C1 was immunoprecipitated using anti-FLAG beads and eluted from the beads using FLAG peptide. Cell extracts and FLAG-eluted proteins (FLAG-IP) were separated by SDS-PAGE, blotted, and probed with anti-HA and anti-myc antibodies.

FIGURE 2.

EIF2C2 is associated with polysomes in HEK293 cells. Cytoplasmic extracts were prepared by cell lysis with buffer containing 5 mM MgCl₂ or 30 mM EDTA, centrifuged through sucrose gradient, and fractionated. Proteins of each fraction were TCA-precipitated and equal aliquots were separated by SDS-PAGE, blotted, and probed with antibodies as indicated. Absorbance of fractions at 254 nm is shown.

FIGURE 3.

EIF2C2 is associated with ribosomes. (A) FLAG/HA-EIF2C2 was immunoprecipitated from a cytoplasmic extract prepared with lysis buffer containing 2 mM EDTA. The epitope-tagged protein was eluted using FLAG peptide and separated on a 10%–45% sucrose gradient for 8.5 h. Proteins of each fraction were TCA precipitated and separated by SDS-PAGE (silver-stained gel is shown on top). Western analysis of the separated fractions was performed using antibodies as indicated. (B) miR-16 is associated with three FLAG/HA-EIF2C2-containing complexes. FLAG/HA-EIF2C2 was purified and displayed on a sucrose gradient as described in A. The distribution of FLAG/HA-EIF2C2 in the gradient was examined by Western analysis using an anti-HA antibody and compared with the presence of RPS6 and RPL7 with antibodies against these ribosomal proteins. RNA was extracted from the material loaded on to the gradient (L), and each fraction of the gradient, pooled as indicated, separated by 15% denaturing PAGE, blotted, and probed for miRNA miR-16. A total of 20 μg of HEK293 cellular RNA (HEK) was loaded along with 10 and 4 fmol of synthetic miR-16 as control.

FIGURE 4.

AGO and TNRC6 proteins immunopurify a similar set of transcripts enriched for sequences complementary to miRNA seeds (A) Two-dimensonal hierarchical clustering for the enriched transcripts in IPs. IPs were performed from extracts of cells stably expressing the FLAG/HA-tagged protein as indicated using anti-FLAG agarose beads (FA) or Protein G Dynabeads bound anti-FLAG (FD), anti-HA (HD), and monoclonal anti-Ago2 (mAD) antibodies with the number of the biological replicates indicated in parentheses. Immunoprecipitated transcripts were defined as transcripts that have a high rank (top 2.5%) in each of the indicated IP experiments. Number of transcripts is shown on the left. The left panel additionally shows seed enrichment (SG1, NSG3) for the transcripts clustered in the right panel. (B) Seed complement enrichment for HEK293 miRNAs in immunoprecipitated transcripts compared with transcripts that were not enriched in the IP. The top 5% most enriched transcripts and random subsets containing the same number of not enriched transcripts obtained from each IPs were scanned for the presence of sequences complementary to the three HEK293 seed groups (SG1–SG3) and the control nonseed group 1–3 (NSG1–NSG3), representing control seed groups of miRNAs not expressed in HEK293 cells. IPs are described in A. (C) Enrichment for conserved seed complements for HEK293 miRNAs in immunoprecipitated transcripts. CSG1 (conserved seed group 1) refers to conserved seed hits of the top five expressed miRNAs. SG1 refers to seed hits of the top five expressed miRNA seed groups without conservation information, and NSG1 and NSG2 represent control miRNA seed groups. (D) Predicting miRNA profiles from AGO-immunoprecipitated transcripts. In the left panel a linear regression model was used to infer the activities of each miRNA seed family (nucleotides 1–8, 133 seed families in total; see Landgraf et al. 2007) based on the representation of transcripts carrying seed matches in AGO-IP. Seed families whose reverse complements are frequent in the immunoprecipitated transcripts receive high activity scores, whereas seed families that do not correlate with the IP data receive low scores. The right panel shows a histogram of the Pearson correlation coefficients resulting from the comparison of the miRNA “activity” predicted by the linear model with each of the measured miRNA expression profiles. The profiles that went into the construction of the histogram are from data of the 177 samples of Landgraf et al. (2007) and the HEK293 profile determined by 454 sequencing in this study (red triangle).

FIGURE 5.

miR-122 transfection and immunoprecipitation of miR-122 mRNA targets. (A) Cells stably expressing FLAG/HA-EIF2C2 were mock transfected and transfected with a miR-122 duplex. Fifteen hours after transfection cells were lysed and the epitope-tagged protein was immunoprecipitated from cytoplasmic extracts with anti-FLAG antibody. RNA was extracted from the cleared cell lysate and the IP. RNA was separated by 15% denaturing PAGE and probed for miR-16 and miR-122. A total of 10 and 4 fmol of synthetic miR-16 and miR-122, respectively, were loaded as standards. The miR-16 blot was reprobed with an oligonucleotide antisense to valine tRNA to ensure equal loading of lysate RNA. (B) Analysis of enriched mRNAs in Ago-IP. The EIF2C2-IP-enriched transcripts were scanned for the presence of sequences complementary to the three HEK293 seed groups (SG1–SG3), the control nonseed groups 1–3 (NSG1–NSG3), and the miR-122 seed in the 3′ UTR. The first and second bar group show the enrichment of complements in immunoprecipitated transcripts of the mock-transfected and mir-122-transfected cells, respectively.

FIGURE 6.

miRNA-mediated repression of 3′ UTRs fused to luciferase reporter genes. Luciferase activity from HEK293 cells cotransfected with each reporter/control psiCHECK construct and 2′-O-methyl oligoribonucleotides antisense to 12 HEK293 miRNAs (red bars) was normalized to that from cotransfection of each reporter construct with 2′-O-methyl oligoribonucleotide antisense to Drosophila miRNA bantam (blue bars). Transfection of parental psiCHECK vector without inserted 3′ UTR (psiCHECK) and 3′ UTR with two artificial binding sites each for miR-16 and miR-196b (psiCHECK T) are indicated. Error bars represent standard deviation of experiment performed with six replicates. Normalized Renilla luciferase (RL) versus firefly luciferase (FL) activities are indicated.