FIGURE 4.

Tethered NOS-3 enhances stability of gfp reporter mRNAs. (A) Experimental design. Reporter mRNAs from dual reporter visualized in dissected gonads (top) with differentially labeled smFISH probes to gfp and mCherry (mCh) mRNAs (bottom). (B) Tethered NOS-3 might increase reporter expression by affecting either stability or translation of the reporter RNA. These two mechanisms have distinct predictions for effects on reporter mRNA abundance. More or less RNA abundance is depicted in the figure as larger or smaller letters, respectively, in animals possessing tethered NOS-3:: λN::FLAG (left column) or untethered NOS-3::FLAG (right column). (C) Representative images of smFISH in wild-type, reporter only, NOS-3::FLAG and NOS-3:: λN::FLAG germlines. Images are average projections of five slices (0.3 µm) from confocal microscopy. gfp mRNAs (gfp smFISH, green), mCherry mRNAs (mCherry smFISH, magenta), GFP protein (fluorescence signal [488], yellow), or DNA (DAPI, light blue). (D) Ratios of gfp:mCherry mRNAs from smFISH quantitation. (***) P-value <0.0001. n.s., not significant (P-value = 0.6352). All reporter strains were significantly different than wild-type (P-value <0.0001). Wild-type (N2, no dual reporter; n = 19), dual reporter only (n = 32), NOS-3::FLAG (n = 37), NOS-3:: λN::FLAG (n = 30) from two combined experiments. (E) Model that NOS-3 directly enhances expression of gld-1 mRNA, perhaps together with the GLD-2 poly(A) polymerase. Repression of FEM-3 is proposed to be indirect through enhancement of an unidentified inhibitory factor.