Extended Data Fig. 10. pUG RNA shortening may act as a brake on TEI.
a, The gel shown is the same as in Fig. 5a, except that oma-1 pUG RNAs from the P0 generation are included for WT and rde-3(−) animals. Data is representative of 3 biologically independent experiments. b, oma-1 pUG RNA reads from MiSeq (n=1 biological experiment) were mapped to oma-1 and the length of the oma-1 mRNA portion of each pUG RNA read was determined (y-axis). Shown is a Box and Whisker plot representing the interquartile range (IQR, box) and median (line in the box) of lengths at the indicated generations after dsRNA treatment. The y-axis starts at the aug of the oma-1 mRNA. The whiskers extend to values below and above 1.5*IQR from the first and third quartiles, respectively. Data beyond the end of the whiskers are outliers and plotted as points. These data support the gel in a, showing that pUG RNAs get shorter in each generation during RNAi-triggered TEI. c, A “ratchet” model to explain pUG RNA shortening. pUG RNA shortening may be due to the 3’→5’ directionality of RdRPs, which, during the maintenance phase of pUG/siRNA cycling (see model in Extended Data Fig. 9), causes each turn of the pUG/siRNA cycle to trigger cleavage and pUGylation of target mRNAs at sites more 5’ than in the previous cycle. Eventually, pUG RNAs are too short to act as RdRP templates, cycling cannot be maintained and silencing ends. Additional support for the ratchet model comes from Fig. 5c, which shows that RNAi-triggered pUG RNAs are longer in MAGO12 mutant animals than in wild-type animals. Note: the P0 generation animals in Fig. 5c were exposed to dsRNA continuously from embryos to adulthood, when they were harvested. These longer pUG RNAs are likely due to continued initiation of pUGylation triggered by the exogenously provided dsRNA without downstream pUG/siRNA cycling. In the absence of this cycling, pUG RNA shortening does not occur. Finally, a number of recent studies in C. elegans have reported transgenerational inheritance of acquired traits, which lasts 3–4 generations69–74. As shown in a, the expression of oma-1 RNAi–directed pUG RNAs also perdures for 3–4 generations. These shared generational timescales of inheritance hint that the inheritance of acquired traits in C. elegans may be mediated by pUG RNAs whose generational “half-life” is limited to 3–4 generations due to the built-in brake on TEI provided by pUG RNA shortening.