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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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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