Posttranscriptional Activation of Gene Expression in Xenopus Laevis Oocytes by Microrna–Protein Complexes (Micrornps)

Posttranscriptional activation of gene expression in Xenopus laevis oocytes by microRNA–protein complexes (microRNPs)

Richard D. Mortensena,1, Maria Serraa,1, Joan A. Steitzb,2, and Shobha Vasudevana,2

aCancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114; and bDepartment of Molecular Biophysics and Biochemistry and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536

Contributed by Joan A. Steitz, April 6, 2011 (sent for review January 5, 2011)

MicroRNA–protein complexes (microRNPs) can activate translation Here, we investigated whether microRNA-mediated activation of target reporters and specific mRNAs in quiescent (i.e., G0) mam- occurs in naturally quiescent-like X. laevis immature oocytes. fi malian cell lines. Induced quiescent cells, like folliculated immature We nd that activation is regulated by the G0-controlling cAMP/ oocytes, have high levels of cAMP that activate protein kinase AII PKAII pathway and identify an endogenous microRNA in the im- (PKAII) to maintain G0 and immature states. We report microRNA- mature oocyte required to increase expression of the cell state regulator Myt1. Thus, microRNA-mediated posttranscriptional up- mediated up-regulated expression of reporters in immature Xen- regulation is relevant for maintenance of the immature oocyte state. opus laevis oocytes, dependent on Xenopus AGO or human AGO2 and on FXR1, as in mammalian cells. Importantly, we find that Results maintenance of cAMP levels and downstream PKAII signaling are Exogenous MicroRNAs Activate Expression of Target mRNA Reporters required for microRNA-mediated up-regulated expression in in the Immature Oocyte. We tested microRNA-mediated expres- oocytes. We identify an important, endogenous cell state regula- sion in the G0-like immature X. laevis oocyte with luciferase tor, Myt1 kinase, as a natural target of microRNA-mediated up- reporters used in mammalian cells (2). We injected DNA con- regulation in response to xlmiR16, ensuring maintenance of oo- structs with a CMV promoter and bovine growth hormone poly- cyte immaturity. Our data reveal the physiological relevance of adenylation sequence or in vitro-transcribed capped, unadenylated cAMP/PKAII-controlled posttranscriptional gene expression activa- RNAs into the nucleus of folliculated stage IV–VI oocytes. We tion by microRNAs in maintenance of the immature oocyte state. routinely included: (i) coinjection of a Renilla luciferase reporter (REN) bearing a nonspecific polylinker sequence in its 3′-UTR to ii fl icroRNAs are 19- to 23-nt RNAs that serve as posttran- normalize for injection and overall translation status; ( ) a Fire y luciferase reporter with either a mutated 3′-UTR or a polylinker Mscriptional regulators of gene expression when recruited ′ into effector complexes with a core Argonaute protein, AGO2 sequence (control; CTRL) of the same size as the 3 -UTR of the – test reporter; and (iii) assessment of RNA levels. (eIF2C2) in mammals. These microRNA protein complexes fl α (microRNPs) bind the target mRNA, normally within its 3′-UTR, When a Fire y luciferase plasmid bearing TNF- AU-rich ele- ments (AREs) in its 3′-UTR was coinjected with the corresponding and regulate translation and decay of mRNAs (1). fi We previously demonstrated that microRNPs can effect microRNA, miR369-3p (2), a ve- to sixfold increase in expression fi was observed relative to the control microRNA, let-7a, or the translation activation of minimal target reporters and speci c A mRNAs in quiescent mammalian cells (2). Quiescence refers to CTRL reporter (Fig. 1 ). An increase in expression can arise from nondividing G0 and G0-like states with specific gene expression relief of repression, whereby the increased expression is equal to programs that dividing cells can enter for extended periods of and not greater than the control (i.e., restoration of general time in a reversible manner. The G0 state can be naturally pro- translation), or activation, in which the increased expression is grammed during differentiation or development or induced in greater than the control (i.e., stimulation over general translation); cultured cells. DNA replication ceases and gene expression skews the control is either a no-microRNA target reporter (i.e., CTRL) or a mismatched control microRNA added with the test reporter toward maintaining the G0 state and preventing promiscuous (2, 10). Expression of the ARE reporter with miR369-3p was entry into other states (3). higher than the reference controls, CTRL and the ARE reporter Like G0 cells, the Xenopus laevis prophase I-arrested immature with a nonmatching microRNA, suggesting stimulation. oocyte does not proliferate or replicate DNA (4). The immature We then injected the CX reporter plasmid, which harbors four oocyte is surrounded by follicle cells that maintain high cAMP copies of a target site for a synthetic microRNA, miRcxcr4, levels and downstream protein kinase A (PKA) signaling, thereby which had previously exhibited translation activation in G0 inhibiting maturation (5). Defolliculation and progesterone mammalian cells and translation repression in cycling cells (2, 11). treatment cause a loss of signaling through G protein-coupled A time course of luciferase activity revealed increased expression CELL BIOLOGY receptors, leading to altered PKA signaling, loss of the nuclear in the presence of miRcxcr4 compared with the control let-7a membrane [called germinal vesicle (GV) breakdown], and mat- microRNA; a fourfold enhancement at 3 h after injection in- uration (5). The cAMP-inducible PKA holoenzyme acts as PKAI creased to sixfold at 5 h (Fig. 1B). Cytoplasmic levels of the mRNA or PKAII as a result of modulation of the catalytic subunit by did not exceed that of the microRNA until 6 h, when the exoge- alternative cofactors, repressor I (RI) or II (RII) subunits (6); nous microRNA levels with a half-life of approximately 30 min both RI and RII respond to cAMP levels, with RII requiring decreased (Fig. S1 A and C and SI Methods). Values increased higher levels of cAMP. PKAI is present in proliferating cells and only for the microRNA target, CX, and not for the control REN various tumors in which RI is overexpressed; PKAII is observed in arrested and nonproliferating cells in which RII predominates (6). Like immature oocytes, the G0 state in some mammalian cells can be elicited by increasing cAMP levels to induce PKAII (6, 7). Author contributions: S.V. designed research; R.D.M., M.S., and S.V. performed research; The oocyte up-regulates the expression of genes essential for R.D.M., M.S., J.A.S., S.V. analyzed data; and J.A.S. and S.V. wrote the paper. maintaining the immature state (8). Among these is the cell state The authors declare no conflict of interest. regulator, Myt1 kinase, which is up-regulated at the translational 1R.D.M. and M.S. contributed equally to this work. level as the immature oocyte advances from stages I–III to stages 2To whom correspondence may be addressed. E-mail: [email protected]. IV–VI (8). Myt1 is required for CDC2 phosphorylation and con- edu or [email protected]. sequent inactivation of prematuration promoting factor (pre-MPF, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. comprised of cyclin B2 and CDC2) (8, 9), preventing maturation. 1073/pnas.1105401108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1105401108 PNAS | May 17, 2011 | vol. 108 | no. 20 | 8281–8286 Downloaded by guest on September 27, 2021 (Fig. S1D), suggesting speciﬁc activation of the microRNA target. Up-Regulated Expression by MicroRNAs Occurs Only in Folliculated The 4-h time point was chosen for comparison in subsequent Immature Oocytes. Up-regulated translation of the CX reporter by experiments. Luciferase protein half-lives (Fig. S1B), CX total miRcxcr4 was observed in folliculated but not in defolliculated (Fig. 1C), and cytoplasmic mRNA (Fig. 1D and Fig. S1C) levels immature oocytes (Fig. 1E). Likewise, when oocytes were ma- were comparable in the presence or absence of miRcxcr4. Com- tured with progesterone, the sixfold stimulation of expression of parable results were obtained by injecting the corresponding the CX reporter was converted to a fourfold repressive effect capped reporter mRNAs and microRNAs into the GV of folli- (Fig. 1F). Thus, maturation, like defolliculation, correlates with a culated oocytes (Fig. 1E). These results suggest that microRNA- loss of up-regulated translation by microRNAs. mediated up-regulated expression at the translational level can be achieved in the natural, G0-like immature oocyte. A

A C

B D

E F

Fig. 2. The cAMP/PKA pathway mediates up-regulated translation by Fig. 1. MicroRNAs mediate up-regulated expression of reporters coinjected microRNPs in oocytes. (A) Addition of 1.25 mM papaverine, a phosphodiesterase into the GV of folliculated stage IV–VI oocytes. (A) MiR369-3p (but not inhibitor that blocks turnover of cAMP, restores up-regulated translation of the control let-7a) up-regulates expression of the ARE reporter (but not of CTRL CX reporter by miRcxcr4 (but not by control miR, let-7a) in defolliculated oocytes. reporter), when coinjected into the GV with the ARE and CTRL luciferase (B) Expression of PKA repressor I (RI) abrogates, whereas repressor II (RII) acti- plasmids along with REN. (B) MiRcxcr4 (but not control let-7a) up-regulates vates, translation of the CX reporter in the presence of miRcxcr4 and of the expression of the CX reporter (2, 11) over time when coinjected with the CX AGO2-tethered reporter in oocytes. (C) Blocking the PKA pathway (dominant luciferase plasmid into GV. MiRcxcr4 and the control miR (let-7a) do not negative PKA, dnPKA) or the downstream PAK pathway (using a dominant alter total oocyte (C) or cytoplasmic levels (D) of the CX reporter mRNA. negative inhibitor, AID) causes loss of up-regulation of CX by miRcxcr4 in folli- Northern blots show the protected 250-nt band for the Firefly luciferase culated immature oocytes compared with a mutant form of the inhibitor, mtAID, reporter and a 100-nt protected band for the coinjected REN reporter or a GFP control. Expression of a constitutively active PAK, L107F, enables up- (SI Methods). (E) MiRcxcr4 (but not control let-7a) up-regulates expression regulated expression of CX by miRcxcr4 in defolliculated oocytes whereas a ki- of the CX transcript when coinjected into GVs of folliculated but not of nase inactive form, K299R, does not. Expression of a constitutively active defolliculated oocytes. (F) MiRcxcr4 represses translation of the CX re- RAFv600E or a constitutively active MEK, MEKDD (both are turned off by PAK), porter in progesterone-matured oocytes as opposed to up-regulated ex- blocks up-regulated expression of CX by miRcxcr4 compared with the GFP control pression in immature, folliculated oocytes. MiR:mRNA base pairing shown in in folliculated immature oocytes. L107F and K299R samples used defolliculated A and B and in E and F, as well as in subsequent figures. C–F used in vitro- oocytes, whereas all other samples were folliculated oocytes. All reporters were transcribed, capped luciferase reporter RNAs. Firefly luciferase values were in vitro-transcribed, capped luciferase RNAs; the signaling proteins were normalized to the cotransfected REN control. expressed from plasmids. Firefly luciferase values were normalized against REN.

8282 | www.pnas.org/cgi/doi/10.1073/pnas.1105401108 Mortensen et al. Downloaded by guest on September 27, 2021 cAMP/PKA Pathway Is Required for Up-Regulated Translation in the active site of PAK (mtAID) does not (Fig. 2C; compare AID Xenopus Oocytes. cAMP levels increase in some G0 mammalian vs. mtAID). In defolliculated oocytes, a constitutively active form cells (6, 7) and in immature folliculated oocytes (Fig. S2A) (5). of PAK (L107F) promotes activation whereas a catalytically in- Maturation can be prevented by adding inhibitors of phospho- active form (K299R) does not (Fig. 2C; compare L107F vs. K299R diesterases and nondegradable cAMP analogues (5). Accord- samples, which are defolliculated oocytes), suggesting that the ingly, the loss of translation activation of the CX reporter by PAK pathway is required for microRNA-mediated up-regulation miRcxcr4 upon defolliculation of immature oocytes can be re- in oocytes. A constitutively active form of RAF, which activates versed by addition of the phosphodiesterase inhibitor papaverine MAPK, or a constitutively active MAPK prevents activation (Fig. (Fig. 2A). Dibutyryl cAMP, an analogue that increases PKA ac- 2C; RAFv600E and MEKDD), arguing that cAMP-mediated activity (12), elicits increased translation of the CX reporter in the tivation by microRNAs involves turning off downstream MAPK presence of miRcxcr4 or of an AGO2-tethered reporter (2) in and involves PAK; additional pathways may also affect activation. defolliculated oocytes (Fig. S2B). In accord with observations of PKAII activity in arrested mammalian cells (6, 7), injection of RII Up-Regulated Expression by MicroRNAs in Immature Oocytes Requires (PKAII activation) or the catalytic subunit (C) plasmids into Human AGO2 or Xenopus AGO and FXR1. We asked whether AGO oocytes activated translation with miRcxcr4 and not with the and FXR1, factors essential for translation activation in G0 control microRNA, let-7a, whereas RI (PKAI activation), asso- mammalian cells (2), are also involved in the oocyte. Western ciated with proliferation, did not (Fig. 2B and Fig. S2C). Over- blotting with an anti-AGO2 antibody as well as an AGO antibody expression of dominant negative PKA inhibited up-regulation in that recognizes AGOs 1 to 4 (Fig. 3A and Fig. S3 A and C) revealed oocytes, conﬁrming that PKA is required for translation activation a band in immature oocyte extracts. cDNA sequencing identiﬁed (dnPKA; Fig. 2C) in immature Xenopus oocytes. an allele, which matched one of two database entries for Xenopus The PKA/cAMP pathway signals to the p21-activated kinase eIF2C2 (NM_001093519) and is similar to a second Xenopus (PAK) to turn off the growth factor-stimulated MAPK pro- eIF2C2 sequence (EU338243). As the cDNA has not been char- liferative pathway in mammalian cells; in oocytes, cAMP and PKA acterized with known human AGO2 functions such as slicer ac- function with PAK to promote maintenance of the immature state tivity (1), we refer to it as xlAGO based on its sequence identity to (13). Injecting a dominant-negative inhibitor of PAK (AID) into the Argonaute family. immature folliculated oocytes prevents translation activation, When oocytes depleted of FXR1 or AGO by antisense for whereas a mutant form of the inhibitor that cannot bind and block 12 to 16 h (Fig. 3A, Fig. S3D,andSI Methods) are injected with

Fig. 3. FXR1 and xlAGO are required for AC microRNA-mediated up-regulated translation in oocytes. (A) Sixteen hours of DNA antisense treatment decreases endogenous FXR1 and AGO protein levels in the oocyte, which were rescued by expression of in vitro-transcribed mRNAs encoding λN-tagged Xenopus FXR1 and AGO, respectively, as observed by Western blotting (SI Methods and Fig. S3D). Actin served as a loading control. Twofold dilutions (12.5–100%) of mock-treated immature oocyte extract (lanes 1–4) are shown: Upper (AGO) and Lower (FXR1). Lanes 5–6 and 7–8(Upper)are duplicate samples, as are lanes 5–7 and 8–9 (Lower). (B) MiRcxcr4 activates translation of D the CX reporter mRNA expressed from a plasmid when coinjected into the nuclei of immature, folliculated oocytes (mock). An- tisense reduction of Xenopus AGO or FXR1 levels abrogates activation of the CX reporter, which can be rescued by expression of the λN-tagged resistant clones. (C)Ex- pression of λN-tagged Xenopus FXR1 or AGO (XlFXR1 and XlAGO) mRNAs into immature oocyte nuclei causes translation ac- CELL BIOLOGY tivation of the coinjected tethered reporter. λN and the λNprpΔ mutant form of human BE AGO2 are controls. (D) Plasmids expressing λN-tagged human FXR1 or AGO2 coinjected into immature oocyte nuclei cause translation activation of the tethered reporter. In vitro-transcribed, capped XlFXR1 and XlAGO mRNAs (A–C), and plasmids expressing hAGO2 and hFXR1 (D) were used for protein expression; experiments in C and D used in vitro-transcribed capped tethered reporter. In B–D, Firefly luciferase values were normalized against REN. (E) RT-PCR analysis (Upper) of RNAs extracted from anti-AGO2 or control anti-FLAG coimmunoprecipitates (Ip) from oocyte nuclear extracts coinjected with the CX or CTRL reporters and the specific miRcxcr4 or the control microRNA, let-7a, reveals significant association of CX but not CTRL mRNA with xlAGO in the presence but not absence of miRcxcr4. (Lower) Western blot of AGO protein in input and immunoprecipitation (Ip) lanes.

Mortensen et al. PNAS | May 17, 2011 | vol. 108 | no. 20 | 8283 Downloaded by guest on September 27, 2021 the CX reporter and the corresponding miRcxcr4, no translation than 6 h for siRNA-mediated antisense depletion, likely because activation is observed (Fig. 3B) compared with a four- to five- of interference with processing or stability instead of cleavage. fold translation up-regulation in mock-treated oocytes. In vitro- Reporters carrying the entire Myt1 3′-UTR or a 528-nt segment transcribed mRNAs of resistant forms of Xenopus FXR1 and bearing the seven putative sites exhibit up-regulated expression in AGO (λNXlFXR1 and λNXlAGO; Fig. 3 A and B) or of human the presence of xlmiR16 (Fig. 4A) independent of RNA levels FXR1 and AGO2 (Fig. S3B) rescue translation, suggesting a (Fig. 4B). We found that one site (1,284 nt from the stop codon) is conserved function between humans and Xenopus; human necessary for up-regulation, as mutating this site (mt3Myt1; SI AGO1, AGO3, and AGO4 have less significant effects on acti- Methods) causes a loss of activation that is not further decreased by vation (Fig. S3B). When λN-tagged human or Xenopus AGO and the loss of xlmiR16 (Fig. 4A). To determine whether the effect of FXR1 are individually expressed along with the 5B Box Firefly xlmiR16 is direct, we made a correspondingly mutated xlmiR16, luciferase tethering reporter, either protein causes translation xlmt3miR16 (SI Methods), which should restore base pairing; we activation, whereas control mutant proteins and vectors do not find that the compensatory microRNA restores translation acti- (Fig. 3 C and D). Finally, to test whether association with the vation (Fig. 4A) without altering mRNA levels (Fig. 4B). mRNA in Xenopus oocytes is microRNA-dependent, we analyzed The xlmiR16 target sequence in the Myt1 3′-UTR defined AGO immunoprecipitates for the Firefly luciferase mRNA by earlier is complementary to nucleotides 4 to 10 (or 4-12) of the RT-PCR. Fig. 3E shows that Xenopus AGO associates signifi- microRNA rather than to nucleotides 2 to 7/8, as expected from cantly with the CX reporter (but not CTRL mRNA) in the the seed rules (15). Therefore, either the Myt1 target site uses presence but not absence of miRcxcr4. These data argue that noncanonical base pairing (16, 17) or the endogenous microRNA Xenopus AGO and FXR1 have retained at least the translation has an altered 5′ end that enables functional interactions (Fig. 4C, activation function of their mammalian counterparts. potential base pairing with Myt1 shown). We determined the 5′ end of the microRNA by splint ligation by using oligonucleo- Endogenous microRNA with a Heterogeneous 5′ End, xlmiR16, Up- tides designed to capture stepwise shortened versions of the Regulates Expression of Myt1 mRNA in the Oocyte. We searched 5′ end of xlmiR16: B1 is the full 5′ end, and B2, B3, and B4 are for potential targets reported to be regulated at the translation successively 1, 2, and 3 nt shorter. The B3 version (truncated by level during oocyte stages III–VI (8). Myt1 protein levels rise 2ntatthe5′ end of xlmiR16) is abundant in both total oocyte RNA across the immature stages without a concomitant increase in and in AGO immunoprecipitates (Fig. 4C), suggesting that B3/ mRNA levels and are required for CDC2 phosphorylation and xlmiR16 is the predominant functional form of the microRNA, inactivation of pre-MPF (8, 9). Myt1 mRNA harbors a 1758-nt 3′- whereas the nontruncated version of xlmiR16 (B1/xlmiR16) is only UTR that contains seven potential xlmiR16 target sites (Fig. 4A) faintly detectable. To prove that B3/xlmiR16 can up-regulate that might engage in noncanonical base pairing. An siRNA an- translation by acting on the full-length Myt1 3′-UTR (potential tisense to the loop region of precursor (pre-) xlmiR16 reduced base pairing with Myt1 shown in Fig. 4C), luciferase assays were mature xlmiR16 levels in oocytes (SI Methods and Fig. S4A). The repeated after depleting endogenous xlmiR16 and adding back endogenous xlmiR16 (14) appears more stable (Fig. S4B) than various 5′-truncated forms. Addition of B3/xlmiR16 restores exogenous microRNAs (Figs. S1A, i, and S4C), requiring more translation activation (Fig. 4D)morerobustlythanB1/xlmiR16.

Fig. 4. Myt1 mRNA translation is up- AC regulated by a 5′ variant xlmiR16. (A) Myt1 3′-UTR has seven putative xlmiR16 sites (dashes). Mutation of the site at 1,284 nt (mt3Myt1 3′-UTR) in the 528-nt or full-length Myt1 3′-UTR failed to demonstrate translation activation and was unaffected by xlmiR16 presence or depletion (SI Methods; siRNAs antisense to pre- miR16 reduced mature xlmiR16 after 6 h, likely because of interference with processing or stability instead of cleavage activity; Fig. S4A). Translation activation is restored by addition of a compensatorily mutated xlmt3miR16 that can base pair to the mutated site (SI Methods). Both B1/xlmiR16 (shown) B and B3/xlmiR16 (Fig. 4C shows base pairing) are able to rescue. (B) North- D ern analyses of Firefly reporters containing the 528-nt Myt1 3′-UTR and mt3Myt1 3′-UTR and REN RNA levels after treatment with si-pre-miR16 with or without mt3xlmiR16 add-back. (Right) Endogenous Myt1 mRNA levels with or without si-pre-miR16 treatment; U6 RNA is a loading control. (C) Splint ligation reactions (SI Methods) containing the labeled 14-nt acceptor oligo and RNAs isolated from anti-AGO2 or -FLAG (control) immunoprecipitates (Ip). XlmiR16 variants that are successively truncated by one nucleotide at the 5′ end (B1/xlmiR16-B4/xlmiR16) were detected using DNA bridge oligos B1-B4, complementary to the first 16 nt of the predicted microRNAs. The predicted full-length form, B1/xlmiR16, is only faintly visible in the sample immunoprecipitated with anti-AGO2, suggesting that it is less abundant than B3/xlmiR16, which is revealed by the B3 bridge oligo to be an abundant microRNA in anti-AGO2 immunoprecipitates. Lanes marked B1/xlmiR16 and B3/xlmiR16 show synthetic miR16 RNAs as size controls. Underlined nucleotides are required for interaction with the Myt1 3′-UTR. Base pairing between Myt1 target site and B1/xlmiR16 or B3/xlmiR16 are shown. (D) Oocytes treated with mock (let-7a) or si-pre-miR16 were coinjected with synthetic xlmiR16 forms to assess rescue of full-length Myt1 3′-UTR reporter translation. Firefly luciferase values were normalized to REN. B1/xlmiR16 undergoes trimming to the B3/xlmiR16 form and rescues translation to a lesser extent than B3/xlmiR16.

8284 | www.pnas.org/cgi/doi/10.1073/pnas.1105401108 Mortensen et al. Downloaded by guest on September 27, 2021 To confirm that the site at 1284 in Myt1 3′-UTR is critical for is rescued by add-back of synthetic xlmiR16 (B3/xlmiR16). Con- translation up-regulation, we designed an LNA oligonucleotide versely, the loss of translation up-regulation observed with the antisense to this site and its adjacent sequences, similar to the mtMyt1 reporter is rescued by addition of a B3/xlmiR16 form, protectors developed earlier (18). When LNAmyt is injected into xlmtmiR16, harboring compensatory mutations that restore base oocytes (Fig. 5A), translation activation of luciferase reporters pairing with the mutant mtMyt1 3′-UTR (Fig. 5A). bearing the 528-nt Myt1 3′-UTR (or full length) is abrogated. Loss of translation activation is also observed when endogenous MicroRNA-Activated Expression Is Required to Maintain the Oocyte xlmiR16 levels are depleted by si-pre-miR16 (Fig. 5A); activation Immature State. Ablation of Myt1 kinase in the oocyte leads to CDC2 dephosphorylation and loss of the immature state (8, 9). We asked whether translation activation via xlmiR16 is essential for maintenance of the immature state. First, we treated oocytes A with the LNA protector, LNAmyt, to block translation activation of the endogenous Myt1 mRNA. As shown in Fig. 5B, increasing levels of LNAmyt reduce endogenous Myt1 protein. Injection of a Myt1 gene construct with a polylinker as its 3′-UTR (Mytctr- lutr), which should not bind LNAmyt, partially restores Myt1 levels (Fig. 5B), demonstrating that the loss of translation activation by LNAmyt is through the Myt1 3′-UTR. Second, we depleted endogenous xlmiR16 levels by using si-pre-miR16 (Fig. 5C) and observed a corresponding loss of Myt1 protein levels (Fig. 5D). We then asked whether xlmiR16-mediated translation activation of Myt1 is physiologically essential by examining the marker of immaturity, phosphorylated CDC2. Phosphorylated CDC2 levels dramatically decrease (Fig. 5E) in concert with the decrease in Myt1 levels. Significantly, addition of synthetic B3/ xlmiR16 (Fig. 5C) restores Myt1 protein levels (Fig. 5D) and phosphorylated CDC2 increases (Fig. 5E). Importantly, loss of MYT1 expression by blocking its 3′-UTR miR16 target site with LNAmyt (Fig. 5B) also leads to loss of immaturity, marked by loss of CDC2 phosphorylation (Fig. S5A, lane 3). Decreased levels of phosphorylated CDC2 and loss of immaturity upon xlmiR16 depletion (Fig. 5E and Fig. S5A, lane 6) or upon LNAmyt protection B C of the miR16 target site on endogenous Myt1 mRNA (Fig. S5A, lane 3) are rescued by injecting the Myt1 construct without its regulatory 3′-UTR (Mytctrlutr; Fig. S5A, lanes 4 and 8; and Fig. S5B). These data argue that xlmiR16 is required to maintain the immature state in part via up-regulating Myt1 expression. Discussion MicroRNPs exhibit versatility in their control of gene expression, D dependent on the specific mRNA 3′-UTR (10, 19). Translational activation in alternative cell states, such as the immature oocyte, E provides a means of gene expression to maintain the state. We previously demonstrated that microRNPs can activate translation of minimal target reporters in G0 mammalian cells (2). Here we have documented microRNA-mediated translation activation in the naturally G0-like X. laevis immature oocyte. xlAGO and FXR1 proteins are required, as in mammalian G0- induced activation. We further demonstrate that microRNA- dependent activation is dependent on cAMP/PKA signaling in Fig. 5. Translation activation of Myt1 mRNA by xlmiR16 is essential for X. laevis oocytes. Our data argue that xlmiR16 is required in part maintenance of the immature state. (A) Coinjection of an LNA antisense for oocyte immaturity and that microRNA-mediated activated oligonucleotide complementary to the xlmiR16 target site (LNAmyt, gray X. laevis ′ expression of endogenous mRNAs in the oocyte is dash) in the 528-nt Myt1 3 -UTR abrogates translation activation, as does physiologically relevant for maintenance of the immature state. knockdown of xlmiR16 with si-pre-miR16, which can be rescued by B1/ A common feature of the immature Xenopus oocyte and CELL BIOLOGY xlmiR16. Mutation of this target site (mtMYT1) abrogates translation acti- mammalian G0 state is that cAMP signaling is required to sustain vation, but add-back of the compensatorily mutated microRNA, xlmtmiR16, immaturity (5, 20) and G0 in some mammalian cells (7). Loss of restores activation. Firefly luciferase values were normalized to REN. Muta- the oocyte immature state leads to loss of microRNA-mediated tions are in lowercase. (B) LNAmyt (increasing twofold, 0.25–4 pmol) blocks E F accumulation of endogenous Myt1 protein in the oocyte, whereas Myt1 activation (Fig. 1 and ), as does loss of the mammalian G0 state protein levels are restored at the highest LNAmyt concentration by coin- (2). cAMP binds the R subunit and activates PKA dependent on jecting a Myt1 expression vector lacking its 3′-UTR (Mytctrlutr), compared the predominance of RI, associated with proliferation or RII, as- with twofold dilutions (25–100%) of control untreated immature oocyte sociated with arrested cells (6). Overexpression of RII/PKAII (but not RI) in oocytes leads to activated expression in the presence of extract. (C) Northern blot demonstrating that depletion of mature xlmiR16 B in the oocyte can be reversed by injecting synthetic mature B3/xlmiR16. U6 microRNAs (Fig. 2 ). PKAII signaling is induced by a relative controlled for loading. (D) Pretreatment with si-pre-miR16, which lowers increase in cAMP levels (21) observed in immature oocytes and endogenous Myt1 protein levels in the oocyte (duplicate lanes), can be re- G0, rather than the total levels of cAMP; therefore, there is a loss versed by injection of synthetic mature B3/xlmiR16 (duplicate lanes) com- of activation controlled by PKAII upon defolliculation (Fig. 1E) pared with untreated immature oocyte extract (100% and 50%). (E) and loss of adenyl cyclase stimulation. However, the effect is more Pretreatment with si-pre-miR16 leads to decreased levels of Myt1 protein pronounced in mature oocytes (5) in which GV breakdown and consequent loss of phosphorylated CDC2, which can be restored by increases phosphodiesterase activity and loss of adenyl cyclase addition of synthetic mature B3/xlmiR16, as judged by comparison with activity and MPF activation perpetuates the decreased cAMP twofold dilutions (25–100%) of untreated immature oocyte extract. Actin response via a feedback loop (5). Stabilized persistence of the low served as the loading control for Western analyses (B, D, and E). cAMP state upon oocyte maturation (5) and in proliferating cells

Mortensen et al. PNAS | May 17, 2011 | vol. 108 | no. 20 | 8285 Downloaded by guest on September 27, 2021 is sufficient for RI/PKA I activity (12, 21), which correlates with 5); Myt1 enables phosphorylation of CDC2, contributing to translation repression (Fig. 2B) after maturation (Fig. 1F). maintenance of the immature state (8, 9); loss of Myt1 translation A second common feature of oocytes and G0 cells is that the leads to maturation (Figs. 5 C–E and Fig. S5 A and B) (9). Oocyte repressive microRNP cofactor GW182 (22) appears compro- immaturity can be rescued in part by restoring xlmiR16 (Figs. mised: GW182 interaction with AGO is not detected in immature 5 C–E and Fig. S5A, lane 7) or by reexpressing Myt1 (Fig. S5 A, or mature oocytes and in mouse oocytes, which fail to show sig- B fi lanes 4 and 8, and ), suggesting that xlmiR16 is necessary for ni cant repression (23), and is reduced in G0 mammalian cells immaturity in part because it maintains the expression of the es- (24), in which activation is observed (2). AGO that cannot in- teract with GW182 can fail to repress translation (23, 25), cause sential regulator, Myt1 (Figs. 4 and 5 and Fig. S5). Other targets activation of unadenylated target reporters, or repress via the cap and (undiscovered) microRNA functions likely also contribute to (26), likely decided by additional determinants. SpecificmRNAs oocyte immaturity. The role of microRNAs in mediating up-reg- may adapt to the absence of the repressive AGO–GW182 in- ulated expression in the oocyte has implications for related phys- teraction in oocytes and G0 (2, 23, 24) and instead recruit iological functions in G0 mammalian cells. a translation activating AGO–FXR1 complex (which relocalizes to polysomes in G0) (2). Regulation by cAMP may give AGO– Methods FXR1 an advantage in oocytes and G0, in which AGO–GW182 Oocytes. Human chorionic gonadotropin (hCG)-stimulated oocytes were interaction is reduced (2, 23, 24), consistent with a recent report manipulated as described in SI Methods. A total of 5 nL containing 0.0625 that FXR1 function in zebrafish requires PAK (27), a kinase to 0.125 ng DNA plasmid, 0.01–0.1 fmol in vitro-transcribed luciferase re- downstream of cAMP required for microRNA-mediated activa- porter mRNAs, 375 fmol siRNA or microRNA, 37.5 ng of antisense or 10 ng tion in oocytes (Fig. 2C). of mRNAs were injected per oocyte with blue dextran dye to visualize The potential mechanisms of enhanced expression, including delivery into the GV. Translation was assayed with a time course ranging mRNA stability and translation up-regulation, remain to be ex- from 1 to 6 h and compared at 4 h in hCG-treated oocytes. All experiments plored. Although the total and cytoplasmic levels of the mRNAs C D C were conducted with folliculated immature stage IV-VI oocytes unless (Fig. 1 and and Fig. S1 ) do not change with the presence of specified otherwise. interacting microRNA, effects of the cap and the poly(A) tail remain to be defined. The experiments were carried out with non- adenylated mRNAs, as enhanced expression compared with the Plasmids and Reporters. All plasmids and reporters are described in SI Methods. increased levels observed with polyadenylated reporters could not be discerned. The need to inject into the nucleus for activation and Luciferase Assay and Protein and RNA Analyses. Luciferase assays were per- the absence of activation in mature oocytes, which lack intact nuclei formed as described (Promega). Oocytes were manually crushed in passive lysis (Fig. 1F), may result from the fact that mRNA translation effects buffer (Promega), and clarified by centrifugation at 2,000 × g for 5 m. Total are often decided by nuclear events (28) and that microRNP oocyte extracts, immunoprecipitations, and RNA analyses are described in SI functions can involve a nuclear phase for AGO (29). Methods. The average ratios of luciferase values were calculated with SDs. MicroRNAs are less abundant relative to other small RNAs in RNAlevelswereassessedineachexperimentseparately(notusedtonor- Xenopus oocytes (14, 30). The range of targets regulated may be malize the values graphed) and performed at least three times completely. expanded by trimming microRNAs in a flexible manner. A 5′- truncated version of B3/xlmiR16 (20 nt) in AGO complexes (Fig. ACKNOWLEDGMENTS. The authors thank Z. Mourelatos, D. Bloch, M. Fritzler, 4C) triggered more robust translation activation of Myt1 mRNA and W. Filipowicz for reagents; P. Vasudevan, K. Tycowski, K. Herbert, A. than the untruncated B1/xlmiR16 (Fig. 4D). Alexandrov, and R.-M. Lye for critical comments; and Y. Tong and S. Truesdell Xenopus for technical and A. Miccinello for editorial assistance. This work was supported oocyte immaturity is maintained by critical phosphor- by a Cancer Research Institute Investigator Award (to S.V.), Leukemia and ylations that render pre-MPF inactive until maturation is induced Lymphoma Society Special Fellowship 3300-09 (to S.V.), Massachusetts General (20). Our results demonstrate that PKA-regulated microRNA- Hospital start-up funds (S.V.), and National Institutes of Health Grants mediated activation is essential for Myt1 expression (Figs. 4 and GM26154 and CA16038 (to J.A.S.).

1. Carthew RW, Sontheimer EJ (2009) Origins and Mechanisms of miRNAs and siRNAs. 15. Bartel DP (2009) MicroRNAs: Target recognition and regulatory functions. Cell 136: Cell 136:642–655. 215–233. 2. Vasudevan S, Tong Y, Steitz JA (2007) Switching from repression to activation: 16. Didiano D, Hobert O (2008) Molecular architecture of a miRNA-regulated 3′ UTR. RNA MicroRNAs can up-regulate translation. Science 318:1931–1934. 14:1297–1317. 3. Coller HA, Sang L, Roberts JM (2006) A new description of cellular quiescence. PLoS 17. Nahvi A, Shoemaker CJ, Green R (2009) An expanded seed sequence deﬁnition Biol 4:e83. accounts for full regulation of the hid 3′ UTR by bantam miRNA. RNA 15:814–822. 4. Taieb F, Thibier C, Jessus C (1997) On cyclins, oocytes, and eggs. Mol Reprod Dev 48: 18. Choi WY, Giraldez AJ, Schier AF (2007) Target protectors reveal dampening and 397–411. balancing of Nodal agonist and antagonist by miR-430. Science 318:271–274. 5. Schorderet-Slatkine S, Schorderet M, Baulieu EE (1982) Cyclic AMP-mediated control 19. Nakahara K, et al. (2005) Targets of microRNA regulation in the Drosophila oocyte of meiosis: Effects of progesterone, cholera toxin, and membrane-active drugs in proteome. Proc Natl Acad Sci USA 102:12023–12028. Xenopus laevis oocytes. Proc Natl Acad Sci USA 79:850–854. 20. Motlík J, Kubelka M (1990) Cell-cycle aspects of growth and maturation of 6. Cho-Chung YS, Nesterova MV (2005) Tumor reversion: Protein kinase A isozyme mammalian oocytes. Mol Reprod Dev 27:366–375. switching. Ann N Y Acad Sci 1058:76–86. 21. Kovo M, et al. (2006) An active protein kinase A (PKA) is involved in meiotic arrest of 7. Friedman DL (1976) Role of cyclic nucleotides in cell growth and differentiation. rat growing oocytes. Reproduction 132:33–43. Physiol Rev 56:652–708. 22. Liu J, et al. (2005) A role for the P-body component GW182 in microRNA function. Nat 8. Furuno N, Kawasaki A, Sagata N (2003) Expression of cell-cycle regulators during Cell Biol 7:1261–1266. Xenopus oogenesis. Gene Expr Patterns 3:165–168. 23. Ma J, et al. (2010) MicroRNA activity is suppressed in mouse oocytes. Curr Biol 20: 9. Nakajo N, et al. (2000) Absence of Wee1 ensures the meiotic cell cycle in Xenopus 265–270. oocytes. Genes Dev 14:328–338. 24. Yang Z, et al. (2004) GW182 is critical for the stability of GW bodies expressed during 10. Bhattacharyya SN, Habermacher R, Martine U, Closs EI, Filipowicz W (2006) Relief of the cell cycle and cell proliferation. J Cell Sci 117:5567–5578. microRNA-mediated translational repression in human cells subjected to stress. Cell 25. Till S, et al. (2007) A conserved motif in Argonaute-interacting proteins mediates 125:1111–1124. functional interactions through the Argonaute PIWI domain. Nat Struct Mol Biol 14: 11. Doench JG, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. Genes Dev 897–903. 17:438–442. 26. Iwasaki S, Tomari Y (2009) Argonaute-mediated translational repression (and 12. Skålhegg BS, Taskén K (1997) Speciﬁcity in the cAMP/PKA signaling pathway. activation). Fly (Austin) 3:204–206. differential expression, regulation, and subcellular localization of subunits of PKA. 27. Say E, et al. (2010) A functional requirement for PAK1 binding to the KH(2) domain of Front Biosci 2:d331–d342. the fragile X protein-related FXR1. Mol Cell 38:236–249. 13. Faure S, et al. (1999) Control of G2/M transition in Xenopus by a member of the p21- 28. Moore MJ, Proudfoot NJ (2009) Pre-mRNA processing reaches back to transcription activated kinase (PAK) family: A link between protein kinase A and PAK signaling and ahead to translation. Cell 136:688–700. pathways? J Biol Chem 274:3573–3579. 29. Weinmann L, et al. (2009) Importin 8 is a gene silencing factor that targets argonaute 14. Armisen J, Gilchrist MJ, Wilczynska A, Standart N, Miska EA (2009) Abundant and proteins to distinct mRNAs. Cell 136:496–507. dynamically expressed miRNAs, piRNAs, and other small RNAs in the vertebrate 30. Lau NC, Ohsumi T, Borowsky M, Kingston RE, Blower MD (2009) Systematic and single Xenopus tropicalis. Genome Res 19:1766–1775. cell analysis of Xenopus Piwi-interacting RNAs and Xiwi. EMBO J 28:2945–2958.

8286 | www.pnas.org/cgi/doi/10.1073/pnas.1105401108 Mortensen et al. Downloaded by guest on September 27, 2021