DEV02655 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 -

  Table S1. Expression of mlt-10p::gfp-pest in let-7(mg279) mir-84(tm1304) adults upon RNAi against genes implicated in molting. Populations of GR1348 let-7(mg279) mir-84(tm1304) mgIs49(mlt-10p::gfp pest) animals were synchronized as L1 larvae, fed E. coli OP50 until the fourth larval stage, and then fed bacteria expressing dsRNA corresponding to the indicated genes. As a control, worms were fed bacteria not expressing dsRNA against a worm gene. Animals were examined for any detectable fluorescence approximately 18 hours later, when the majority of control animals expressed GFP. Values represent the combined results from two independent experiments. Clones that yielded 80% normalized fluorescence with a P-value >0.001 were tested again (not shown). Only RNAi of nhr-25 significantly suppressed (P′′0.001) both GFP expression and lethality in the additional test.

• Supplemental Table 2 -

  Table S2. Viability of let-7(mg279) mir-84(tm1304) mutant adults following inactivation of genes implicated in molting. Viability of the identical animals described in Table S1 was assessed by visual inspection approximately 45 hours after transfer to E. coli expressing dsRNA. Values for particular gene inactivations represent the combined results from two independent experiments. In this table, values reported for vector 1 and vector 2 include the combined results from multiple independent samples reported separately in Table S1 as, respectively, vector 1, 2, 3 and 4, or vector 5 and 6. Note that RNAi of a very small number of these genes, including Y37D8A.21, did not reproducibly interfere with molting of larvae, and those particular genes were therefore not discussed in Frand et al., 2005 (Frand et al., 2005).

• Supplemental Figure 1 -

  Fig. S1. Models of base-pairing between let-7 paralogs and the transcripts of alg-1 and pan-1. These targets were identified on the basis of forming an unbroken helix with the 5′ end of the miRNA (at least bases 2-7) and the absence of G:U base-pairs in this ‘seed’ sequence. The pairing with the most favorable predicted free energy of folding is depicted. (A) Base-pairing between the alg-1 3′ UTR and mir-48. The same site is also predicted to bind let-7, mir-84 and mir-241. The target site in alg-1 corresponds to nucleotides 3440 through 3466 of cosmid F48F7 (accession number, emb|Z69661). (B) Base-pairing between the pan-1 3′ UTR and mir-241. The same site is also predicted to bind mir-84 and mir-48. The target site in pan-1 corresponds to nucleotides 32,352 through 32,380 of cosmid M88 (accession number, emb|Z34802).

• Supplemental Figure 2 -

  Fig. S2. Reporters for mir-84 show dynamic expression during larval development. (A) The percentage of animals that expressed mir-84::gfp in a given tissue at each stage is indicated. (B) An adult showing mir-84::gfp expression in the pharynx, vulva (arrowhead) and spermathecae (arrows). (C) mir-84::gfp expression in the somatic gonad commenced in the L2 stage. (D) A L3 animal expressing mir-84::gfp in the anchor cell and vulval precursor cells. (E) A L4 animal showing vulval mir-84::gfp expression in the descendants of P5.p and P7.p. (F) A mir-84::yfp reporter bearing 8.1 kb of promoter sequence was expressed in the same tissues as mir-84::gfp. Shown here is an L3 animal expressing mir-84::yfp in the pharynx, anchor cell (arrow) and Pn.p cells (arrowheads), some of which are the vulval precursor cells.