Molecular Machinery Involved in the Process of Small RNA Mediated Gene Regulation

Micro-RNA (miRNA) and small interfering-RNA (siRNA) are short (~22-nucleotide), single-stranded RNA molecules that regulate gene expression by promoting degradation or translational inhibition of target mRNAs. They influence diverse biological functions through the repression of target genes during normal development and pathological responses. A hallmark of small-RNA mediated gene silencing is a class of approximately 22-nucleotide RNAs that are processed from double-stranded RNA precursors by Dicer. The current model suggests that Dicer selects cleavage sites by measuring a set distance from the 3′ overhang of the double-stranded RNA terminus. Our structural studies on human Dicer in complex with siRNA having different overhang lengths at 5’- and 3’-ends along with in vitro and in vivo functional studies showed that Dicer recognizes both 3’- as well as 5’-ends for proper cleavage of siRNA/miRNA. The 5'-end recognition by Dicer, demonstrated for the first time by us, is important for precise and effective biogenesis of siRNA/miRNA. This study has provided practical benefits to the design of small-RNAs for gene silencing. We also solved the crystal structure of a large complex of Trax–translin heteromers, also known as C3PO, which has been proposed to activate the RNA-induced silencing complex by facilitating endonucleolytic cleavage of the siRNA passenger strand. Our studies establish that Trax adopts the translin fold, possesses catalytic centers essential for C3PO's endoribonuclease activity and interacts extensively with translin to form an octameric assembly. This study provides important insights into the catalytic mechanism of C3PO and its conserved role in human RISC activation. Just as miRNAs and siRNAs bind to the Argonaute proteins, another class of small RNAs encoded in the genome, the Piwi-interacting RNAs (piRNAs), that are 2′-O-methylated at their 3′ ends, bind to the Piwi proteins. Germline-specific piRNAs and Piwi proteins play a critical role in genome defense against transposable elements. Our work on Piwi proteins demonstrated the structural basis for piRNA 2'-O-methylated 3' end recognition by the PAZ domain of Piwi proteins.