Figure 1. Isolation of Deletion Alleles of the
let-7 Family MicroRNA Genes
mir-48,
mir-84, and
mir-241. (A) Alignment of mature 22 nt microRNA sequences of the four C. elegans
let-7 family members and the human
let-7 RNA (hs
let-7). Shaded boxes and asterisks indicate bases conserved in two or three worm family members, respectively.(B) Temporal expression patterns of
let-7 family microRNA genes,
mir-48,
mir-84,
mir-241, and
let-7. Northern blot analysis of RNA isolated from populations of staged worms. As a control for staging of the worms during early larval development,
lin-4 expression is observed during the late L1 stage. Northern blots indicate that miR-241, miR-48, and miR-84 reached half-maximal expression at about the L3 stage, while
let-7 RNA reached half-maximal expression at about the L4 stage. Each blot was stripped and probed for U6 snRNA expression to standardize loading of RNA samples. The numbers underneath each band indicate the signal of the corresponding microRNA normalized to that of U6 snRNA presented relative to maximal expression for each microRNA.(C) List of deletion mutations isolated in the
mir-48,
mir-84, and
mir-241 genes.(D) Northern blot analysis of RNA isolated from wild-type or mutant animals and hybridized with probes to the mature 22 nt microRNA sequences for miR-48, miR-241, and miR-84. The deletion mutations result in the loss of the corresponding 22 nt mature microRNA for miR-48, miR-241, and miR-84.(E) The
mir-48 and
mir-241 locus on chromosome V. The mature microRNA sequences are located within a 1.8 kb genomic region on cosmid F56A12. Deletions at this locus are shown below. The deficiency nDf51 removes both the
mir-48 and
mir-241 mature microRNA sequences. For rescue experiments, we injected the plasmid pAAS50, which contains a 5073 bp fragment of genomic DNA encompassing both the
mir-48 and
mir-241 mature microRNA sequences. For control experiments, we injected the plasmid pAAS60, which was generated from pAAS50 but lacked the sequences corresponding to the
mir-48 and
mir-241 mature microRNA sequences.