Fig 2: Nuclear localization of Dicer and Ago2. (A) Western analysis of Dicer in cytoplasmic and nuclear fractions. The 5% cytoplasmic and 20% nuclear fractions prepared from HeLa cells were loaded in a 4–12% SDS–PAGE gel, transferred to a membrane and proteins were detected using antibodies. Alpha-tubulin and hnRNP A2 were used as controls for cytoplasmic and nuclear proteins, respectively. (B) Ago2 can be found in both cytoplasmic and nuclear fractions. Western analysis was performed using the same samples as in (C), and the membrane was probed using different antibodies. GAPDH and hnRNPA2 were detected and used as controls for cytoplasmic and nuclear proteins, respectively. (C) Localization of Dicer (upper panel) and Ago2 (lower panel) in Hela cells. Cells grown on glass-bottom dishes were fixed, stained with first antibodies against Dicer (1:200, ab14601, from mouse, Abcam) and Nucleolin (1:200, ab22758, from rabbit, Abcam), or against Ago2 (1:150, ab57113, from mouse, Abcam) and Nucleolin, as described in ‘Materials and Methods’ section. Secondary antibodies were anti-mouse antibody (1:200, ab6785, Abcam, conjugated with FITC (green) and anti-rabbit antibody (1:200, Ab6719, conjugated with Texas Red). Nucleolin (red) was detected and served as a nucleolar marker. The nucleus was stained with DAPI (blue). The arrow indicates the positions of nucleoli. The scale bars: 10 µm.

Fig 3: 3-Cyanovinylcarbazole-based miRNA target pulldown. (A) CNVK mimic-based microRNA target pulldown methodology. (B) MTT assay-based assessment of NIH3T3 cell viability measured 24 h after transfection with CNVK-miR-29b. (C) TaqMan qRT-PCR based assessment of miR-29b expression (normalized to U6 expression) in untransfected cells and cells transfected with increasing doses of CNVK-miR-29b and CNVK-scram. (*) P < 0.05 compared to CNVK-scram; two-tailed Student's t-test. (D) Representative immunoblot demonstrating association of CNVK-miR-29b and CNVK-scram with Argonaute2 protein in HeLa cells. Of note, 1/20th volume of the irradiated cell lysate was loaded in the Input lanes. Remaining lysate was affinity purified using streptavidin magnetic beads and all of it was used for the Pulldown lanes. GAPDH is used to evaluate nonspecific protein pulldown. The antibody used for HeLa lysate is anti-human a-AGO2 antibody (ab57113, Abcam). M refers to molecular weight marker (kDa). (E) NIH3T3 cells were transfected with 20 nM concentration of CNVK-miR-29b, CNVK-scram (negative control oligo), n-miR-29b, or C. elegans miR-67 (negative miRNA control) oligos, and gene expression values were normalized to Hprt1. Gene expression levels in CNVK-miR-29b transfected cells are plotted as a ratio of respective gene levels in CNVK-scram transfected cells (set at one), and gene expression values in n-miR-29b transfected cells are reported as a ratio of expression values in C. elegans miR-67 transfected cells (set at one). Irf6 is used as nontarget control gene. (*) P < 0.05 compared to respective negative control oligos; two-tailed Student's t-test. qRT-PCR analysis of previously reported miR-29b target gene expression in NIH3T3 cells transfected with 10 nM CNVK-miR-29b, bio-miR-29b, or CNVK-scram oligonucleotides. Transfected cell lysates were irradiated with 365 nm UV light for either (F) 10 min or (G) 20 min to induce covalent crosslinking of CNVK with complementary nucleotides in target mRNA. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001 compared to other transfected cells; one-way ANOVA, followed by Tukey's post hoc test. Bars represent mean ± SEM of three independent experiments.

Fig 4: Differential RISC association of endogenous miRNAs in 293 cells. (A and B) Illumina deep sequencing was performed for either RISC-associated miRNAs, isolated by immunoprecipitation using either the diagenode 2A8 or Abcam ab57113 pan-Ago specific monoclonal antibody, or Total small RNAs (≤200 nt). Raw reads were aligned to mature human miRNAs and normalized to total miRNA-aligned reads. Individual miRNAs that represented >0.1% of either miRNA population were then raised to log base 2 and plotted as percent miRNA reads in the eluate versus percent reads in the total miRNA pool. Lines indicate a 2-fold difference from the mean. miRNAs highlighted in grey were verified by qRT-PCR (see below), while miRNAs indicated by name were used for functional assays. (C) qRT-PCR was performed using Taqman probes for miR-101-3p, miR-22-3p, miR92–3p and miR-197-3p using the same RNA preparations used for deep sequencing in panels A and B. (†) Note that the Taqman probe used to measure miR-92a-3p levels will also detect miR-92b-3p. However, both of these closely related miRNAs are predicted to be equally overrepresented in RISC (panels A and B).

Fig 5: Schematic diagram for eIF1A functions in Ago2-dependent miR-451 biogenesis, RNA interference and erythrocyte maturation in zebrafishThe primary miRNA (priRNA) is cleaved by Drosha-DGCR8 pathways to generate pre-miRNA within the nucleus. Exportin-5 in complex with Ran-GTP exports the pre-miRNA to the cytoplasm, where the pre-miRNA is bound by DICER to form a RISC Loading Complex that includes Ago2. In the canonical miRNA biogenesis pathway, DICER removes the terminal loop region to yield the mature miRNA. The pre-miR-451 is loaded directly onto Ago2 and sliced on the 3′ hairpin arm. The miR-144 biogenesis is DICER-dependent. The globular domain (GD) of eIF1A directly binds to MID-domain of Ago2 and forms an eIF1A-Ago2 complex promoting Ago2-mediated RNAi and miR-451 biogenesis. MID-domain of Ago2 binds to the GD of eIF1A and does not impair eIF1A functions in translation initiation. The 5′-term of guide strand of miRNA-miRNA* duplex is docked onto a pocket with residues mainly from MID-domain. The long and structured mRNAs are scanned by RISCs to recognize seed regions in miRNAs. After perfect or imperfect complementary guide with miRNAs, mRNAs are nicked by PIWI domain resulting in RNAi. eIF1A augments Ago2-mediated RNAi, miR-451 production and erythrocyte maturation. Black arrows are from previous reports of other groups14,15,43; blue arrows are from this study.