Abstract Post-transcriptional gene silencing (PTGS) mediated by small silencing RNAs such as ~22-24 nt microRNAs (miRNAs) and ~21 nt small interfering RNAs (siRNAs) produced by Dicer enzymes, is important in every aspect of biology. Making small RNAs with an appropriate length is crucial for their functions. Small RNAs with incorrect lengths, which can be produced if Dicer selects incorrect cleavage sites in precursor RNAs, can cause detrimental effects in cells. Thus, understanding the molecular mechanisms by which the length of small silencing RNAs produced by Dicer are determined and regulated is significant and is the goal of research in this proposal. Drosophila Dicer-1 makes ~22-24 nt miRNAs from pre-miRNAs while Dicer-2 precisely produces ~21 nt siRNAs from long dsRNAs derived from virus and transposon. We recently reported that the miRNA length produced by Drosophila Dicer-1 and mammalian Dicer can be changed by its binding partner proteins Loquacious-PB (Loqs-PB) in Drosophila and TRBP in mammals, respectively, but not by their alternative partners Loqs-PA and PACT. However, the molecular mechanism by which Loqs- PB/TRBP, but not Loqs-PA/PACT, changes the length of miRNAs is unknown, and we will aim to better understand it. We will test the hypothesis that Loqs-PB/TRBP, but not Loqs-PA/PACT, binds the unpaired bases at the central part of the pre-miRNA stem and changes their conformation in order to change the miRNA length. In contrast to miRNAs that have a broader length distribution (~22-24 nt), the length of ~21 nt siRNAs is more precise. The difference in the length between miRNAs and siRNAs is important for their respective functions. However, how Drosophila Dicer-2 can produce siRNAs with such high precision is unknown. Based on our previous and preliminary results, we will test the hypothesis that the phosphate-binding pocket in the PAZ domain of Dicer-2 plays crucial roles to produce precise ~21 nt siRNAs. We also hypothesize that the C-terminal dsRNA-binding domain (dsRBD) of Dicer-2 additionally contributes to the precision of the ~21 nt siRNA production. We hypothesize that the phosphate-binding pocket and C-terminal dsRBD anchors the terminal monophosphate and body of long dsRNAs, respectively, thereby aligning the RNAs precisely along the RNaseIII active site. To achieve these aims, we will use Drosophila genetics, reconstituted in vitro biochemistry, and high-throughput sequencing. The proposed studies will reveal the molecular mechanisms by which the length of miRNAs and siRNAs are defined and regulated, which will advance our understanding of sequence-specific post-transcriptional gene silencing.