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. 2017 May 30;114(24):6310–6315. doi: 10.1073/pnas.1610417114

Fig. S2.

Fig. S2.

Tether-function reporter mRNAs are stable in oocytes. (A) Diagram of reporter and internal control mRNAs used in the tether-function assays throughout this report. ICP27 is expressed in X. laevis stage VI oocytes as a fusion with the phage MS2 coat protein (MS2) and tethered to luciferase mRNA via the interaction of MS2 with three cognate-binding sites within the 3′ UTR (Luc-MS23). Reporter mRNAs are unadenylated, unless otherwise stated ([3], [4]), and have a 5′ m7GpppG or ApppG cap (m7G-Luc-MS23 [1], [3] and ApG-Luc-MS23 [2], [4]) or an IRES as indicated (CSFV-Luc-MS23, PV-Luc-MS23, or HAV-Luc-MS23 [5]) in the main text. IRES-reporter constructs are ApppG-capped. A luciferase reporter mRNA lacking the MS2-binding sites is designated ApG-Luc-ΔMS2 [6]. An m7GpppG-capped polyadenylated lacZ mRNA containing no MS2-binding sites is used as an internal control (β-gal [7]). For the modified tether-function assay (Figs. 4C and 5B), an ApppG-capped, unadenylated, CSFV IRES-dependent internal control mRNA (CSFV–β-gal [8]) was used. (B and C) ICP27 alone is insufficient to activate VP16 mRNA translation efficiently. (B) X. laevis oocytes were injected with 50 ng of mRNA expressing ICP27, and ICP27 protein was detected by immunoblotting after overnight incubation. (C) In vitro-transcribed mRNA encoding VP16 (50, 250, or 500 pg) was injected into oocytes expressing either ICP27 or U1A (negative control), and VP16 protein was detected by immunoblotting after overnight incubation. (D) ICP27 does not affect reporter mRNA levels, consistent with the remarkable stability of mRNAs in oocytes (18). Oocytes expressing MS2 or MS2-ICP27 were injected with ApG-Luc-MS23, PV-Luc-MS23, HAV-Luc-MS23, or CSFV-Luc-MS23 reporter mRNAs. Total RNA was extracted either immediately (0h) or 14 h (14h) after injection and analyzed by quantitative RT-PCR with primers against the luciferase ORF, showing equivalent levels of mRNA. The number of amplification cycles required to obtain a detectable product in the linear range (threshold cycle) is shown. Error bars represent SEM.