Journal: Nucleic Acids Research

Article Title: Genome-wide analysis reveals a switch in the translational program upon oocyte meiotic resumption

doi: 10.1093/nar/gkaa010

Figure Lengend Snippet: The presence of CPEs in the 3′UTR is associated with translational repression in prophase I-arrested oocytes. ( A ) TE values of members of the Oosp cluster during meiosis. The average and range of TEs are reported. ( B ) Polyadenylation state of members of the Oosp cluster in Pro I-arrested oocytes. Data were from a published TAIL-Seq study . ( C ) Accumulation of YPet reporters for Oosp1 and Oosp2 3′UTRs during meiotic maturation. Pro I-arrested oocytes were collected and microinjected with oligoadenylated YPet-Oosp1 (red) or YPet-Oosp2 (blue) mRNA along with polyadenylated mCherry mRNA. Oocytes were allowed to recover for 16 h after microinjection, released from cilostamide, and imaged for 16 h with a sampling frequency of 15 min. Each point is the mean ± SEM of individual oocyte traces obtained in three separate experiments. The total number of oocytes analyzed is in parentheses. ( D ) YPet reporters for Oosp1 and Oosp2 3′UTRs. 3′UTRs expressed in the oocytes were cloned downstream of the YPet ORF (yellow box). CPEs (gray ovals) and PASes (green hexagons) are shown along with nucleotide positions relative to the start of the 3′UTR. ( E ) Accumulation of Oosp1 and Oosp2 3′UTR YPet reporters in Pro I-arrested oocytes. Pro I-arrested oocytes were collected and microinjected with oligoadenylated YPet-Oosp1 (red) or YPet-Oosp2 (blue) mRNA along with polyadenylated mCherry mRNA. Oocytes were allowed to recover for 2.5 hrs after microinjection, maintained in Pro I, and imaged for 9 h with a sampling frequency of 15 min. Each point is the mean ± SEM of individual oocyte traces obtained in three separate experiments. The total number of oocytes analyzed is in parentheses. ( F ) Translation rates of the Oosp1 and Oosp2 YPet reporters in Pro I-arrested oocytes. The translation rate for each oocyte was calculated by linear regression of the reporter data (E) between 6 and 9 h. Mean ± SEM is reported. Statistical significance was evaluated by Mann–Whitney test; **** P < 0.0001. ( G ) Translation rates of oligoadenylated and polyadenylated Oosp1 YPet reporters in Pro I-arrested oocytes. Experimental conditions were as described in (E). The data were collected from two independent experiments and the total number of oocytes analyzed and mean ± SEM are reported. Statistical significance was evaluated by Kruskal?–Wallis test; **** P < 0.0001 and ns: not significant. ( H ) Translation rates of oligoadenylated or polyadenylated Oosp2 3′UTR YPet reporter in Pro I-arrested oocytes. Pro I-arrested oocytes were collected and microinjected with either YPet-Oosp2-oligo(A) or YPet-Oosp2-poly(A) mRNA along with polyadenylated mCherry mRNA. Experimental conditions were as described in (E). The translation rate for each oocyte was calculated by linear regression of the reporter data between 0 and 3 h or 6 and 9 h. The data were collected from two independent experiments and the total number of oocytes analyzed and mean ± SEM are reported. Statistical significance was evaluated by Kruskal–Wallis test; **** P < 0.0001 and ns: not significant. ( I ) Mutations of CPE(s) in the Oosp1 YPet reporter. The proximal site is designated as CPE1 and the distal as CPE2. CPE1 (TTTTAAATaaa) was mutated to ‘CGACAAATaaa,’, preserving the downstream, overlapping PAS, while CPE2 (TTTTAAT) was mutated to ‘CGACTCC’ as previously described . ( J ) Accumulation of wild type Oosp1 , wild type Oosp2 , and mutant Oosp1 reporters in Pro I-arrested oocytes. Pro I-arrested oocytes were collected and microinjected with oligoadenylated YPet-Oosp1 (red circle), YPet-Oosp2 (blue circle), YPet- Oosp1(ΔCPE1) (red square), YPet-Oosp1(ΔCPE2) (red triangle) or YPet-Oosp1(ΔCPE1+2) (red diamond) mRNA along with polyadenylated mCherry mRNA. Experimental conditions were as described in (E). Each point is the mean ± SEM of individual oocyte traces obtained in two separate experiments. The total number of oocytes analyzed is in parentheses. ( K ) Translation rates of wild type Oosp1 , wild type Oosp2 and mutant Oosp1 reporters in Pro I-arrested oocytes. The translation rate for each oocyte was calculated by linear regression of the reporter data (J) between 6 and 9 h. Mean ± SEM is reported. Statistical significance was evaluated by Kruskal–Wallis test; **** P < 0.0001 and ns: not significant. ( L ) Accumulation of YPet-Oosp1 in Pro I-arrested CPEB1 +/+ , CPEB1 +/− and CPEB1 −/− oocytes. Oocytes were collected from hormone-primed wild type, Zp3-Cre T Cpeb1 F/+ , and Zp3-Cre T Cpeb1 F/F mice. Experimental conditions were as described in (E). Each point is the mean ± SEM of individual oocyte traces obtained in two separate experiments. The total number of oocytes analyzed is in parentheses. ( M ) Translation rates of YPet-Oosp1 in Pro I-arrested CPEB1 +/+ , CPEB1 +/− and CPEB1 −/− oocytes. The translation rate for each oocyte was calculated by linear regression of the reporter data (L) between 6 and 9 h. Mean ± SEM is reported. Statistical significance was evaluated by Kruskal–Wallis test; ** P = 0.0043 and ns: not significant.

Article Snippet: The assays used were: Astl (Mm00553165_m1), Bcl2l10 (Mm00478988_m1), Ccnb1 (Mm03053893_gH), Cdk8 (Mm01223097_m1), Depdc7 (Mm00522683_m1) Dnmt (Mm01151063_m1), Dppa3 (Mm01184198_g1), Ewsr1 (Mm01191469_g1), Ing3 (Mm00458324_m1), Mos (Mm01700521_g1), Nlrp5 (Mm01143609_m1), Obox5 (Mm00773197_gH), Oosp1 (Mm00504796_m1), Oosp2 (Mm03015599_m1), Padi6 (Mm00462201_m1), Smc4 (Mm00713073_m1), Tcl1 (Mm00493475_m1), Tiparp (Mm00724822_m1), Zp1 (Mm00494367_m1), Zp2 (Mm00442173_m1) and Zp3 (Mm00442176_m1).

Techniques: Microinjection, Sampling, Clone Assay, MANN-WHITNEY, Preserving, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Genome-wide analysis reveals a switch in the translational program upon oocyte meiotic resumption

doi: 10.1093/nar/gkaa010

Figure Lengend Snippet: CPEB binding to mRNAs activated during maturation is necessary, but not sufficient, for full translational activation ( A ) Pattern of ribosome loading onto UP mRNAs during meiotic maturation. mRNAs whose translation increased by at least 3-fold from Pro I to Met I in our RiboTag/RNA-Seq dataset are shown. Traces of the 149 mRNAs with the highest activation are in grey and transcripts recovered in the pellet of RNA-IP/RT-qPCR with CPEB1 antibody are in black. * denotes transcripts that are also immunoprecipitated by DAZL antibodies (data under review). ( B ) RiboTag-IP/RT-qPCR validation of ribosome loading for selected UP candidates. Zp3-Cre T RiboTag F/F mice were hormone primed and the oocytes isolated. Oocytes were either maintained in Pro I or matured in vitro for 8 h and collected for downstream RiboTag-IP/RT-qPCR analysis. We quantified several candidates with some of the greatest fold changes in ribosome loading from Pro I to Met I. Dppa3 was used as a reference gene as it is known to be constitutively translated during this time. Data are represented as fold changes in message levels as compared to 0 h. Three biological replicates of 200 oocytes per time point were used and RT-qPCR reactions were run in triplicate. The bars represent the mean ± SEM of three experiments. Statistical significance was evaluated by unpaired, two-tailed t -tests; **** P < 0.0001. ( C ) The effect of CDK1 inhibition on the translation of Ccnb1 mRNA (UP). Pro I-arrested oocytes were collected and microinjected with oligoadenylated YPet-Ccnb1 3′UTR mRNA along with polyadenylated mCherry mRNA. Oocytes were incubated for 16 h then two groups of oocytes were maintained in Pro I with either cilostamide (empty, black circle) or dinaciclib without cilostamide (blue circle). Another two groups of oocytes were either matured without (solid, black circle) or with dinaciclib added at 2 h after release (red circle). Imaging started 2 h after cilostamide release and lasted for 10 h with a sampling frequency of 15 min. Each point is the mean ± SEM of individual oocyte traces obtained in three separate experiments. The total number of oocytes analyzed is in parentheses. ( D ) Translation rates of YPet-CcnB1 and YPet-Ewsr1 are affected by CDK1 inhibition during meiotic maturation. The translation rate for each oocyte was calculated by linear regression of the reporter data (C and ) between 8 and 12 h. Mean ± SEM is reported. Statistical significance was evaluated by Kruskal–Wallis test; ns: not significant; **** P < 0.0001. ( E ) Detailed analysis of the relationship between mRNAs that are translationally activated during meiotic resumption and the presence of CPEs in the 3′UTR. Pie charts report the percentage of UP mRNAs in Pro I-arrested oocytes that have or lack CPEs in the 3′UTR. ( F ) CPEB1 is required for efficient translational activation of CcnB1. CPEB1 +/+ (black), CPEB1 +/− (light red) and CPEB1 −/− (red) oocytes were collected, maintained in Pro I, and microinjected with oligoadenylated YPet-CcnB1 mRNA along with polyadenylated mCherry mRNA. After 2.5 h incubation, oocytes were matured and imaged for 10 h with a sampling frequency of 15 min. Each point is the mean ± SEM of individual oocyte traces obtained in two separate experiments. The total number of oocytes analyzed is in parentheses. ( G ) Translation rates of the YPet-CcnB1 reporter during oocyte maturation in CPEB1 +/+ , CPEB1 +/− and CPEB1 −/− oocytes. The translation rate for each oocyte was calculated by linear regression of the reporter data (F) between 0 and 2 h or 6 and 10 h. Mean ± SEM is reported. Statistical significance was evaluated by Kruskal–Wallis test; ns: not significant; **** P < 0.0001. ( H ) Accumulation of wild type Oosp1 and mutant Oosp1 YPet reporters during meiotic maturation. Pro I-arrested oocytes were collected and microinjected with oligoadenylated YPet-Oosp1 (circle), YPet-Oosp1(ΔCPE1) (square), YPet-Oosp1(ΔCPE2) (triangle) or YPet-Oosp1(ΔCPE1+2) (diamond) mRNA along with polyadenylated mCherry mRNA. After 16 h of recovery after microinjection, oocytes were allowed to mature, and imaged for 10 h with a sampling frequency of 15 min. Each point is the mean ± SEM of individual oocyte traces obtained in two separate experiments. The total number of oocytes analyzed is in parentheses. ( I ) Translation rates of wild type Oosp1 and mutant Oosp1 YPet reporters during meiotic maturation. The translation rate for each oocyte was calculated by linear regression of the reporter data (H) between 0 and 2 h or 6 and 10 h (post-GVBD). Mean ± SEM is reported. Statistical significance was evaluated by Kruskal–Wallis test; ns: not significant; **** P < 0.0001.

Article Snippet: The assays used were: Astl (Mm00553165_m1), Bcl2l10 (Mm00478988_m1), Ccnb1 (Mm03053893_gH), Cdk8 (Mm01223097_m1), Depdc7 (Mm00522683_m1) Dnmt (Mm01151063_m1), Dppa3 (Mm01184198_g1), Ewsr1 (Mm01191469_g1), Ing3 (Mm00458324_m1), Mos (Mm01700521_g1), Nlrp5 (Mm01143609_m1), Obox5 (Mm00773197_gH), Oosp1 (Mm00504796_m1), Oosp2 (Mm03015599_m1), Padi6 (Mm00462201_m1), Smc4 (Mm00713073_m1), Tcl1 (Mm00493475_m1), Tiparp (Mm00724822_m1), Zp1 (Mm00494367_m1), Zp2 (Mm00442173_m1) and Zp3 (Mm00442176_m1).

Techniques: Binding Assay, Activation Assay, RNA Sequencing, Quantitative RT-PCR, Immunoprecipitation, Biomarker Discovery, Isolation, In Vitro, Two Tailed Test, Inhibition, Incubation, Imaging, Sampling, Mutagenesis, Microinjection