FIGURE 1.

Tethered NOS-3 increases GFP reporter expression. (A) Previous tethering assay used in the C. elegans germline (Aoki et al. 2018, 2021). GFP reporter mRNA is under control of a germline-expressed, mex-5 promoter and has three boxB stem–loops in its 3′UTR. The RNA-binding protein (RBP) is tagged with λN. (B) Dual reporter tethering assay (this work). The nascent transcript driven by mex-5 promoter is resolved by trans-splicing into two mRNAs that encode distinct reporters. The gfp reporter RNA has three functional boxB stem–loops in its 3′UTR; the mCherry reporter 3′UTR has three mutated boxB stem–loops that do not bind λN and therefore provides an internal control. Addition of an OLLAS tag to GFP and a V5 tag to mCherry enables sensitive immunostaining and immunoblotting. (C) Schematic of C. elegans germline. Germline stem cells (GSCs) reside in the progenitor zone (yellow); GSC daughters enter and progress through meiotic prophase (green) and finally differentiate into oocytes (pink); red box, mid-pachytene region imaged in Figure 1D; bracket, region quantitated in several figures begins at distal end and goes 310 µm, stopping near the proximal end of the pachytene region. (D) Confocal images (max projection) of germlines stained to detect NOS-3::FLAG and NOS-3:: λN::FLAG (αFLAG, column 1); GFP::H2B::OLLAS::PEST (αOLLAS, columns 2 and 3); V5 antibodies to detect mCherry::H2B::V5::PEST (αV5, column 4), and DNA (DAPI, column 5). All images were acquired with the same microscope settings. All images in each column were visualized at the same contrast in Image J; the lower OLLAS contrast in column 2 was optimized for reporter expression with NOS-3:: λN::FLAG, while the higher constrast in column 3 was optimized for expression with the dual reporter only and NOS-3::FLAG. Wild-type has no tagged proteins and serves as a negative control. Note that images for wild-type, reporter only, NOS-3::FLAG and NOS-3:: λN::FLAG are replicated in Figure 3B. (E) In situ quantitation of GFP (αOLLAS) normalized to mCherry (αV5) reporter proteins, as a function of germline position. Bold lines show averages, and dashed lines and shading show one standard deviation (see Materials and Methods). Wild-type serves as a negative control. Wild-type (N2, no dual reporter; n = 54), NOS-3::FLAG (n = 34), NOS-3:: λN::FLAG (n = 52) worms were analyzed for D and E. Same curves and values are included in Figure 3D; Supplemental Figure S1D,E and Supplemental Figure S2. (F) Immunoblot to assay GFP (αOLLAS) and mCherry (αV5) reporter protein expression. Actin serves as a loading control, and wild-type worms as the negative control (last lane). Whole worm lysate (n = 60 young adult worms) used for all samples.