Supplemental Figures - Journal of Cell Science · PDF file 2016-03-08 ·...

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  • J. Cell Sci. 129: doi:10.1242/jcs.179218: Supplementary information

    Supplemental Figures

    Fig. S1. Cell specific pull-down of ribosome associated mRNAs during oocyte maturation.

    A - Oocytes were collected from Zp3-Ribotag and control mice and used for SDS-PAGE

    immunodetection of proteins. HA – hemagglutinin Ribotag; β-tub – Beta tubulin. B - Ovaries

    were collected from Zp3-Ribotag fixed and HA expression assessed by immunofluorescence.

    HA was detected in oocytes from growing follicles (AF), and transitional follicles (TF) but not

    in primordial follicles (PF). The positive control Vasa denotes germ cells. C and D -

    Microarray analysis of polysome bound messages during oocyte maturation recovered from

    polysome fractionation by sucrose density gradient. Dppa3 shows a constitutive recruitment of

    ribosomes through meiosis (C), whilst Tex19.1 progressively increases recruitment throughout

    the same period (D) GV – germinal vesicle; GVBD – germinal vesicle breakdown; MII –

    second meiotic arrest.

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  • J. Cell Sci. 129: doi:10.1242/jcs.179218: Supplementary information

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    Fig. S2. Translation of Tex19.1 is dependent on cell cycle progression. Schematic diagram

    depicting the structure of a Renilla luciferase reporter with the 3’ UTR of Tex19.1, marking

    important cis elements (A) together with the schematic procedure for oocyte micro-injection and

    IVM (B). C - GV mice oocytes were collected and injected with a luciferase reporter with the 3’

    UTR of Tex19.1. Oocytes were then incubated in media containing a meiotic resumption inhibitor

    or in inhibitor free media. A time course was built by collecting oocytes every two hours.

    Reporter luciferase signal was normalised to the firefly luciferase injection control. D – Nucleotide

    sequence of different luciferase reporters with the 3’ UTR of Tex19.1 with mutated putative

    motives. IVM – in vitro maturation; D – putative DAZL binding motif; ncCPE – Putative non-

    consensus cytoplasmic binding element; cCPE – consensus CPE; Δ – adenosine mutagenesis of

    RBP binding sites.

  • J. Cell Sci. 129: doi:10.1242/jcs.179218: Supplementary information

    Fig. S3. RBPs depletion prevents progression to MII. A – GV oocytes were injected with RBP

    specific morpholino oligonucleotides (MO) (CPEB1 – A; or DAZL – D) or control-MO, left to

    recover 24 hours in meiotic inhibitor media and matured for 5 hours. SDS-PAGE

    immunodection with specific antibodies shows a marked knockdown (KD) for both RBPs,

    CPEB1 and DAZL. Following KD of specific CPEB1 or DAZL oocytes were transferred to

    meiotic inhibitor free media and maturation rate access after 17hrs. C and F - qPCR analysis of

    endogenous messages following CPEB1 and DAZL KD by MO show no affect in the total

    amount of message between different groups. (RNA levels measured on input samples

    representing 10% of the RIP sample in Fig 2A and B). WT – wild type; Het - DAZL+/-; Ctr-MO

    – control morpholino; GV – germinal vesicle; GVBD – germinal vesicle breakdown; MII –

    second meiotic arrest. Jo

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  • J. Cell Sci. 129: doi:10.1242/jcs.179218: Supplementary information

    Fig. S4. Computational analysis of the rate of translation of Tex19.1 Rluc reporters. A -

    Time course of wild type Tex19.1 RLuc reporter translation (Data from Fig. 1E). Data between 0

    and 8 hrs are reported and fitted with the exponential equation Y = Y0(k.X) where Y0 is the initial

    Y value (expressed as ratio RLuc/FLuc), k is the rate constant (expressed as 1/hr) and Tau is the

    reciprocal of k. The R square is reported as a measure of the goodness-of-fit. Linear regression

    gives considerably lower R square values (0.77) than fitting with an exponential equation and

    non-linear regression. B and C - Time course of Tex19.1 RLuc reporter in cells depleted of

    CPEB1 (C; data from Fig. 3A) or time course of ΔCPE Tex19.1 RLuc reporter (B; data from

    Fig 5A). Best fit for these data is again obtained with an exponential equation. D and E - Time

    course of Tex19.1 RLuc reporter in cells depleted of DAZL (D; Data from Fig 3B) or DAZL Δ1

    +3 Tex19.1 RLuc reporter (E; data from Fig 6B). Data can be equally fitted with a linear or

    exponential equation with comparable R square. F - Maximal rates of reporter translation were

    calculated for all the curves and are plotted starting from the same X=0. An additional set of data

    is included where the rate of translation of a WT Tex19.1 RLuc reporter in oocytes that are

    maintained in GV is calculated. This measurement provides a minimal rate of reporter

    translation in the absence of any activation. Note that either RBP depletion or mutation of the

    3’UTR binding site causes similar decreases in maximal rates of translation of the reporter.

    Disruption of CPEB1 activity reduces the rate of translation significantly less that DAZL

    manipulation. The rate of translation of the DAZL Δ1+3 mutant is not significantly different

    from the background rate of translation in oocytes arrested in GV, suggesting that this mutation

    is sufficient to prevent any form of translational activation.

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