Extended Data Fig. 1: Optimization and validation of the suntag-nanos system.
From: Direct observation of translational activation by a ribonucleoprotein granule
a, Schematic of CRISPR knocked-in suntag-nanos allele. b, Images of germplasm in embryos expressing suntag-nanos and (top) scFv-sfGFP (super-folder GFP) or (bottom) monomeric msGFP2 (green). Suntag mRNA is stained by suntag probes (magenta). ScFv-sfGFP showed puncta of GFP signals (arrowheads) which are not co-localized with mRNA signal and thus are not translating sites. ScFv-msGFP2, which is used throughout this study, strongly reduces the aggregation. c, The percentage of bright GFP foci co-localized with mRNA. Large aggregates of scFv-sfGFP colocalize rarely with RNA (~30%) (n = 3 embryos). With scFv-msGFP2, the majority of GFP foci are associated with RNA (80%-90%) and most likely represent polysomes (n = 7 embryos). Data are the mean ± s.d. d, Embryos expressing suntag-nanos and scFv-GFP (green) treated with 20 mM HEPES (control), 10 mg/ml puromycin, or 100 µg/ml harringtonine. Treated embryos were aged for at least 15 min before fixation and staining with suntag probes (magenta).
