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Genes Cells
2018 May 01;235:332-344. doi: 10.1111/gtc.12580.
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MicroRNP-mediated translational activation of nonadenylated mRNAs in a mammalian cell-free system.
Wakiyama M, Ogami K, Iwaoka R, Aoki K, Hoshino SI.
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MicroRNAs are small noncoding RNAs that regulate translation and mRNA stability by binding target mRNAs in complex with Argonaute (AGO) proteins. AGO interacts with a member of the TNRC6 family proteins to form a microRNP complex, which recruits the CCR4-NOT complex to accelerate deadenylation and inhibits translation. MicroRNAs primarily repress translation of target mRNAs but have been shown to enhance translation of a specific type of target reporter mRNAs in various experimental systems: G0 quiescent mammalian cells, Xenopus laevis oocytes, Drosophila embryo extracts, and HeLa cells. In all of the cases mentioned, a common feature of the activated target mRNAs is the lack of a poly(A) tail. Here, we show let-7-microRNP-mediated translational activation of nonadenylated target mRNAs in a mammalian cell-free system, which contains over-expressed AGO2, TNRC6B, and PAPD7 (TUTase5, TRF4-1). Importantly, translation of nonadenylated mRNAs was activated also by tethered TNRC6B silencing domain (SD), in the presence of PAPD7. Deletion of the poly(A)-binding protein (PABP) interacting motif (PAM2) from the TNRC6B-SD abolished the translational activation, suggesting the involvement of PABP in the process. Similar results were also obtained in cultured HEK293T cells. This work may provide novel insights into microRNP-mediated mRNA regulation.
Figure 1. Let‐7 microRNAs stimulate the translation of nonadenylated target mRNAs in a mammalian cell‐free system. Two different m7G‐capped mRNAs, encoding Renilla luciferase (RLuc‐6xT) and firefly luciferase (FLuc), were simultaneously translated in a cell‐free system in the absence and presence of let‐7 microRNA‐like siRNAs. (a) HEK293F cells were transfected individually with an expression vector for FLAG‐tagged AGO2, GW182, TNRC6B, PAPD7, and prepared extracts from each of the cell lines. Cell extracts were analyzed by Western blotting using the antibodies indicated. M: Magic mark XP (Thermo Fisher Scientific); 1: wild‐type HEK293F cells; 2: AGO2; 3: GW182; 4: TNRC6B; 5: PAPD7; 6: wild‐type HEK293F cells; 7: wild‐type HEK293F cells; 8: PAPD7; 9: wild‐type HEK293F cells. The positions of the full‐length GW182 and TNRC6B proteins are marked by arrows. (b) Schematic representations of the RLuc‐6xT and FLuc mRNAs are shown on the top. Measurements of dual‐luciferase assay after 180 min of translation are shown (n = 3, dark, intermediate, and light gray bars; average, blue bar). The cell‐free systems are composed of extracts from wild‐type HEK293F cells and those expressing FLAG‐tagged AGO2, GW182, TNRC6B, and PAPD7 (where indicated). *corresponds to p‐value < .01 obtained with a two‐tailed unpaired Student's t‐test.
Figure 2. Let‐7 microRNAs induce deadenylation but activate translation of target mRNAs in the presence of PAPD7. Cell‐free translation reactions were carried out in the presence of FLAG‐tagged AGO2 and TNRC6B. PAPD7 was included in the indicated experiments. Note dilution rate of the samples subjected to dual‐luciferase assays was different from that of the experiments in Figure 1. (a) Time‐course measurement of RLuc activity produced from RLuc‐6xT(A31) mRNAs. (b) Time‐course measurement of FLuc activity. (c) Poly(A) status of RLuc mRNAs in panel (a) at the time point of 180 min, according to 3′‐end linker ligation and RT‐PCR analysis. The length of the PCR products produced from deadenylated mRNAs is shorter than 300 bp. (d) Measurements of dual‐luciferase assay after 180 min of translation are shown (n = 3, dark, intermediate, and light gray bars; average, blue bar).
Figure 3. PAPD7 impacts translation of polyadenylated mRNAs and PABPC1‐tethered mRNAs. (a) The m7G‐capped RLuc‐5boxB mRNA and m7G‐capped FLuc mRNA were simultaneously translated in a cell‐free system containing lambdaN‐HA‐tagged PABPC1 (PABP) and FLAG‐tagged PAPD7 as indicated. Measurements of dual‐luciferase assay after 195 min of translation are shown (n = 3, dark, intermediate, and light gray bars; average, blue bar). (b) FLAG‐SBP‐tagged PAPD7 mixed with a carboxyl‐terminal FLAG‐tagged PABPC1 construct (schematically shown on the right) was pulled down with streptavidin‐beads. Captured proteins were detected by Western blotting using an anti‐FLAG M2 antibody. PAPD7 interacts with PABPC1‐FLAG and PABPC1(RRM1‐4)‐FLAG but not with PABPC1(C‐term)‐FLAG. M: Magic mark XP (Thermo Fisher Scientific).
Figure 4. Tethered TNRC6B silencing domain (6B‐SD) activates translation of nonadenylated mRNAs in the presence of PAPD7. (a) Schematic representations of TNRC6B protein, 6B‐SD, and 6B‐SDΔP are shown. (b) Deletion of the PAM2 motif from TNRC6B‐SD specifically abrogates interaction with PABPC1. HEK293T cells were transfected with the indicated plasmids. Cell extracts were used for immunoprecipitation using anti‐HA antibody in the presence of RNase If followed by Western blotting with antibodies against the proteins indicated on the left. (c) Time‐course measurement of RLuc synthesis in the cell‐free system containing FLAG‐tagged PAPD7. Nonadenylated & m7G‐capped RLuc‐5boxB mRNA and m7G‐capped RLuc‐5boxB‐poly(A) mRNA (poly(A) length, ~260 nucleotides) were added as indicated. LambdaN‐HA‐tagged proteins tethered to the RLuc mRNAs (6B‐SD, 6B‐SDΔP) are indicated (black line with triangles: no tether). (d) Nonadenylated RLuc‐5boxB mRNA and FLuc mRNA were translated in the absence and presence of FLAG‐tagged PAPD7, lambdaN‐HA‐tagged 6B‐SD, or 6B‐SDΔP as indicated. Measurements of dual‐luciferase assay after 180 min of translation are shown (n = 3, dark, intermediate, and light gray bars; average, blue bar). (e) Polyadenylated (~260 nucleotides) RLuc‐5boxB‐poly(A) mRNA and FLuc mRNA were translated in the absence and presence of FLAG‐tagged PAPD7, lambdaN‐HA‐tagged 6B‐SD, or 6B‐SDΔP as indicated. Measurements of dual‐luciferase assay after 180 min of translation are shown (n = 3, dark, intermediate, and light gray bars; average, blue bar). (f) Poly(A) status of RLuc mRNAs in panel (c) at the time point of 180 min, according to 3′‐end linker ligation and RT‐PCR analysis. The mRNAs tethered to lambdaN‐HA‐tagged 6B‐SD or 6B‐SDΔP were deadenylated.
Figure 5. PAPD7 switches the function of TNRC6B‐SD from translational repression to translational activation. HEK293T cells were transfected with plasmids expressing lambdaN‐HA‐tagged TNRC6B‐SD (6B‐SD), TNRC6B‐SDΔP (6B‐SDΔP), FLAG‐tagged PAPD7, PAPD7(DADA), and the corresponding empty vectors (–) as indicated; 42 hr later, NLuc‐5boxB and FLuc (A31 tail) control mRNAs were transfected for 6 hr. (a) Western blot analysis using the indicated antibodies. (b) Reporter expression levels analyzed by dual‐luciferase assay. NLuc values were normalized to FLuc values, and the normalized levels in empty plasmid‐transfected cells were set to 1. Data are means ± 1 SD (n = 3). Asterisks denote significant difference (p‐value < .01) from the empty‐vector‐transfected cells obtained with a two‐tailed unpaired Student's t‐test.
Figure 6. A model for microRNP‐mediated mRNA regulation in concert with PAPD7. (a) PAPD7 somehow represses translation of polyadenylated mRNAs. (b) TNRC6B, recruited by AGO2, causes PABP dissociation from the mRNA poly(A) tail, and elicits deadenylation. PABP, bound to TNRC6B, changes its conformation when released from poly(A). (c) TNRC6B‐PABP complex activates translation of nonadenylated mRNAs in concert with PAPD7.
