1. miRNA Reading: http://www.ambion.com/techlib/hottopics/rnai/ Lecture notes

2. Components of the RNAi machinery are used for other processes.

3. The first miRNA was discovered in C. elegans. lin-14(lf) mutations caused precocious development. lin-14(gf) and lin-4 mutations caused retarded development. wild type: embryo L1 L2 L3 L4 adult preccious: embryo L2 L3 L4 adult retarded: embryo L1 L1 L2 L3…..

4. lin-14(lf); lin-4(lf) mutants are precocious.

5. lin-14 gene (simplified) loss-of-function gain-of function mutations in coding sequences mutations in 3’UTR lin-4 encodes no protein. Only an RNA. LIN-14 RNA always present, but protein decreases through larval development. lin-4 required for LIN-14 decrease. lin-4 RNA increases though larval development. lin-4 RNA potentially could base pair with lin-14 3’ UTR.

6. lin-4 lin-14 Developmental time LIN-14 protein LIN-14 protein in wild type LIN-14 protein in lin-14(gf) or lin-4(lf) lin-4 RNA

7. Model

8. lacZ lin-14 3’UTR Used lac-z::lin-14 3’ UTR reporter transgene to test model LacZ decreases through larval development. lin-4 required for LacZ decrease. Reduction of Dicer and Risc components results in lin-4 phenotype; also required for LacZ decrease.

9. miRNA complex contains Risc proteins

11. Two Drosophila miRNAs regulate cell proliferation, apoptosis and fat storage.

12. ~300 estimated miRNAs in C. elegans ~530 estimated miRNAs in humans