Figure 6
Regulation of target genes by C. elegans mir-228 and mir-235. (a) Levels of predicted target genes T04A8.7, H19N07.4 (mboa-2), F22A3.1 (ets-4) and F33D4.6 were assessed relative to constitutively expressed C. elegans actin (act-3). Data are expressed as Log2 fold change in mutant strain CLB061 (mir-235(n4504); mir-228(n4382)) relative to N2 wild-type strain. PCR reactions were carried out in triplicate and the mean of three biological replicate RNA samples from synchronized L4 is shown +/− standard deviation. (b) Repression of firefly luciferase in HEK293 cells transfected with the 3′ UTR of predicted targets of cel-mir-235 (F33D4.6, ets-4, zmp-2). Data are expressed as the mean ratio of three experiments comparing Firefly to Renilla luciferase signal in the presence of cel-mir-235 cloned in the forward orientation relative to the reverse orientation, where the reverse is expressed as 1. Error bars indicate standard deviation (two-tailed t-test *P = 0.045 with F33D4.6 3′UTR and P = 0.044 for ets-4 3′UTR). (c) Percentage of adult worms expressing GFP in posterior gut nuclei of strain RAF1713(rrrSi400 [Pets-4::ets-4::gfp::ets-4 3′UTR] II.; unc-119(ed3) III) [71] or strain CLB072 (two independent lines 1 and 2), generated by crossing RAF1713 with MT17997(mir-235(n4504)I. Error bars indicate standard deviation (two-tailed t-test, P = 0.022 and P = 0.047 for CLB072 lines 1 and 2, respectively). (d) ets-4::GFP expression in posterior gut nuclei in CLB072 but not in RAF1713, indicating enhanced GFP expression in the absence of cel-mir-235. Images taken at same exposure at x40 magnification. To enable visualisation of GFP expression, RNAi of ets-4 suppressor rege-1 was required38 and was carried out by RNAi feeding of both RAF1713 and CLB072 strains, in an identical manner. Arrow indicates rectum, arrowheads indicate GFP+ gut nuclei. Smaller autofluorescent gut granules can also been seen.
