Truesdell SS , Mortensen RD , Seo M , Schroeder JC , Lee JH , LeTonqueze O , Vasudevan S .

	Figure 1. Upregulated expression requires injection of the microRNA and target mRNA into the nucleus.(a) Nuclear injections (black bars) demonstrate upregulated expression of the CX reporter with miRcxcr4 and not with control let-7a while cytoplasmic injections demonstrate modest repression (gray bars).Titrating the reporter (nuclear injections at 0.3 fmols or 0.03 fmols of reporter mRNA, shown 0.03 fmols) maintains activation (injections performed with RNA as depicted above38); cytoplasmic injections (at 0.03 fmols of reporter mRNA (shown) or lower at 0.001 fmols-0.0001 fmols or higher upto 3.3 fmols) show no significant changes while increasing the injected microRNA levels does not stimulate38. MicroRNA half-lives and export were demonstrated previously38 (b) mRNA levels of the CX reporter normalized to Renilla reporter levels as analyzed by qRT-PCR do not reflect the changes observed in the translation levels with nuclear or cytoplasmic injections. (c) AGO2 and control RPA antibody immunoprecipitates (Ips, Western blots shown below) from nuclear or cytoplasmically injected (N-inj or C-inj, injected with CX reporter, Renilla reporter and miRcxcr4) oocytes were analyzed by qRT-PCR for the target CX reporter and control Renilla reporters compared to 10% Input samples. Approximately 25% of CX reporter is detected associated with AGO2 in the nuclear injected oocytes compared to 4% in the cytoplasmically injected oocytes. The average values of three replicates with standard deviations as error bars are shown in a-c.
	Figure 2. Xenopus AGO and an endogenous microRNA, miR16, are substantially present in the nucleus in immature oocytes.All extracts (RNA and protein) and nucleus/cytoplasm isolation were prepared as described3738 (Methods). (a) Western analysis of total (T), nuclear (N) and cytoplasmic (C) sonicated extracts from 20 immature, folliculated oocytes using histone H4 as a marker for nuclei and tubulin as a cytoplasmic marker. A lane was left after each fraction loaded to preclude signals from spillover (observed with the tubulin signal in the lane between the cytoplasm and total samples). AGO is more clearly detected when cytoplasmic extracts are sonicated (shown) than in soluble cytoplasmic extracts (all other figures)38. (b) The antibody used for AGO analysis, detects expression from Flag-tagged Xenopus AGO and human (hAGO2) clones. In vitro transcription coupled translation RRL extracts (Promega) were used to express control GFP, Flag-tagged Xenopus AGO and human AGO2 (hAGO2) that were further subject to Flag purification and subsequent Western analyses with the AGO2 antibody used (Millipore, AGO2 antibody) and cross-checked with Flag antibody. (c) Northern analysis of endogenous miR16 in nuclei and cytoplasm of immature oocytes using probes against U6 as a nuclear marker and 5.8S rRNA as a cytoplasmic marker. Endogenous miR16 is substantially nuclear. (d) Splint ligation reactions for detection of miR16/B3 5′ trimmed form using a specific B3 or a control bridging oligonucleotide (bridge splint) as described previously38 with nuclear and cytoplasmic oocyte RNA samples from 20 oocytes each. In vitro synthesized miR16/B3 form was used as a positive control and tRNA as a negative control.
	Figure 3. Upregulated expression requires injection of the microRNA into the nucleus.(a) Nuclear injections (black bars) of miRcxcr4 but not control let-7a (miRcontrol) with cytoplasmic injection of the CX and Renilla reporters rescues upregulated expression of the CX reporter unlike cytoplasmic injections of miRcxcr4, where modest repression is observed (gray bars); injections were performed with transcribed RNA as depicted above.(b) mRNA levels of the CX reporter normalized to Renilla reporter levels as analyzed by qRT-PCR with nuclear or cytoplasmic injections of the microRNA do not correlate with translation changes. The average values of three replicates with standard deviations as error bars are shown in a-b.
	Figure 4. Myt1, an endogenous target mRNA for activation by miR16, associates with AGO in the nucleus where AGO interacts substantially with FXR1, a co-factor required for activation.(a) Immunoprecipitation of Xenopus AGO (anti-AGO/2A8 antibody39) or control RPA from nuclear extracts followed by RT-PCR analysis demonstrated that Myt1 mRNA was specifically associated with AGO in the nucleus; Cyclin A1 mRNA, which is repressed in immature oocytes32 and tRNAlys served as negative controls to test specificity.(b) Immunoprecipitation of AGO (anti-AGO/2A8 antibody39) or control (RPA antibody) from nuclear and cytoplasmic extracts followed by Western blot analyses using anti-AGO2 (Millipore) and anti-FXR1 (Abcam). AGO immunoprecipitates demonstrated association with FXR1 in both the nucleus and cytoplasm, with more substantial co-immunoprecipitation of FXR1 with AGO in the nucleus, indicating the presence of a prominent nuclear AGO-FXR1 complex.
	Figure 5. Expression of FXR1-iso-a to overexpress FXR1 levels increases the cytoplasmic levels of AGO and rescues translation activation of CX mRNA by miRcxcr4, injected cytoplasmically into oocytes.(a) Oocytes were nuclear injected with DNA plasmids to express either GFP control or FXR1-iso-a in oocytes and were subsequently cytoplasmically injected with miRcxcr4 or control let-7a, CX and Renilla reporters.Upregulated expression of the CX reporter in the presence of miRcxcr4 was rescued upon overexpression of FXR1-iso-a but not with GFP control or with the control microRNA. (b) Western blot analysis of nuclei (N) and cytoplasm (C) extracts (soluble extracts that were not sonicated) from 20 immature, folliculated oocytes each that were injected with DNA plasmids to express either GFP control or FXR1-iso-a. Histone H4 and actin served as controls. FXR1-iso-a expression leads to increased FXR1 levels as well as increased cytoplasmic levels of AGO. AGO is more clearly detected when cytoplasmic extracts are sonicated (Fig. 2a) compared to soluble cytoplasmic extracts (this figure)38. (c) mRNA levels of the CX reporter normalized to Renilla reporter levels as analyzed by qRT-PCR with GFP or FXR1-iso-a expression do not correlate with translation changes in (a). (d) AGO2 (Wako) immunoprecipitates from cytoplasmic samples expressing either GFP control or FXR1-iso-a were analyzed for co-immunoprecipitation of FXR1. Increased levels of FXR1 were immunoprecipitated with samples overexpressing FXR1-iso-a. RPA antibody immunoprecipitation served as an antibody control. Increased AGO and FXR1 levels can be observed in the Input lane of the samples overexpressing FXR1-iso-a compared to Actin levels used as a loading control; the AGO antibody amounts (1/3) used for immunoprecipitation are limiting. The average values of three replicates with standard deviations as error bars are shown in a, c.
	Figure 6. AGO2, FXR1 and microRNAs like miR16 are present in mammalian nuclei and are partially restricted by LMB inhibition of the CRM1 export pathway.(a) Western analysis of AGO2 and FXR1 in nuclear and cytoplasmic extracts from proliferating (Cycling) and G0 THP1 cells that were untreated or treated (+)with LMB to inhibit the CRM1 pathway849.CBP80 served as a marker for nuclei and tubulin as a cytoplasmic marker. (b) MicroRNAs are present in the nucleus and cytoplasm as previously published82330445760. MiR16 levels in proliferating (non-G0) and G0 cell nuclei and cytoplasm as analyzed by qRT-PCR. The average values of three replicates with standard deviations as error bars are shown.
	Figure 7. AGO2 interacts substantially with FXR1 and associates with activated but not repressed targets in G0 human cell nuclei.All interactions were observed after in vivo formaldehyde crosslinking4054 followed by nuclear-cytoplasmic fractionation and immunoprecipitation. (a) Immunoprecipitation of AGO2 (Wako) or control IgG from nuclear and cytoplasmic extracts of proliferating (Cycling) and G0 THP1 cells followed by RT-PCR analysis demonstrated that TNFα mRNA was specifically associated with AGO2 exclusively in G0 nuclei after 4 hrs of serum-starvation induction of G0 (TNFα, first panel, 4 hrs G0) and also in the G0 cytoplasm later at 8 hrs of serum-starvation (TNFα, third panel, 8 hrs G0). Cyclin E mRNA, which is repressed in G034, served as a control and is not present in the nuclear complex but in the G0 cytoplasmic complex (Cyclin E, second panel, 4 hrs G0; the mRNA is downregulated and below detection by 8 hrs). Input (INP) = 10% of the sample. (b) G0 HEK293 cells were transfected with reporters that bear the AU-rich target sequence of TNFα mRNA (TNFα ARE, sufficient for activation in G05455) or a mutated sequence (mtARE, non-functional for activation)5455. Immunoprecipitation of AGO2 or control IgG from nuclear and cytoplasmic extracts from transfected late G0 cells followed by RT-PCR analysis demonstrated that the ARE reporter was associated substantially with AGO2 in G0 nuclei, with some association in G0 cytoplasm as expected at late G0, similar to (a). No association was observed with the mutated (mtARE) reporter; tRNAlys and Renilla served as controls. The average values of three replicates with standard deviations as error bars are shown. (c) Western analyses of immunoprecipitates from 4hr G0 and cycling (Cyc) samples from (a) using anti-AGO2 (Millipore) and anti-FXR1. Input (INP) = 10% of the sample. A non-specific band immunoprecipitated with anti-AGO2 and IgG in the AGO2 Western blot analysis below the specific AGO2 band. Anti-FXR1 recognizes several bands in the Input lane representing seven isoforms and modifications. In G0 cytoplasmic fractions (Cyto-G0), the antibody immunoprecipitated less AGO, possibly due to the epitope being masked. In cycling cell cytoplasmic fractions (Cyto-Cyc), AGO is immunoprecipitated at equal levels to that in the nucleus but FXR1association is clearly reduced. AGO2-FXR1 complex is substantially present in the nucleus.
	Figure 8. Expression of FXR1-iso-a to overexpress FXR1 levels increases the cytoplasmic levels of AGO2 and rescues translation activation of CX mRNA by miRcxcr4 in asynchronous, low density proliferating cells.(a) Western analysis of nuclear and cytoplasmic extracts from asynchronous, low density proliferating HEK293 cells (slowly proliferating, mostly lag phase cells) that express either GFP control or FXR1-iso-a (FXR1, 1µg transfected/ 1x105 cells/ml).FXR1-iso-a overexpression leads to increased FXR1 levels as well as increased cytoplasmic AGO2 levels with some increase in the nucleus (short exp = short exposure of the saturated levels present in the original exposure above). Histone H4 and tubulin served as nuclear and cytoplasmic markers, respectively. (b) Immunoprecipitation of AGO2 from cytoplasmic extracts expressing FXR1-iso-a or GFP in asynchronous, low density proliferating cells demonstrated increased interaction between AGO2 and FXR1-iso-a upon FXR1-iso-a but not GFP expression. (c) Expression of either GFP control or FXR1-iso-a in asynchronous, low density proliferating cells that were subsequently transfected with miRcxcr4 or let-7a (control), CX and Renilla (Ren) reporters, demonstrated rescued upregulated expression of the CX reporter in the presence of miRcxcr4 upon overexpression of FXR1-iso-a but not GFP control. The fold activation depicted (fold activation = the Firefly Luciferase translation value of CX reporter observed in the presence of miRcxcr4 normalized to Renilla over the Luciferase value of CX with control Let-7a microRNA normalized to Renilla that were further normalized to their RNA levels, which do not change significantly as described previously)54 are compared between samples overexpressing GFP or FXR1-iso-a. The average values of three replicates with standard deviations as error bars are shown.

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