RNAi revised--target mRNA-dependent enhancement of gene silencing.

The discovery of RNA interference (RNAi) gave rise to the development of new nucleic acid-based technologies as powerful investigational tools and potential therapeutics. Mechanistic key details of RNAi in humans need to be deciphered yet, before such approaches take root in biomedicine and molecular therapy. We developed and validated an in silico-based model of siRNA-mediated RNAi in human cells in order to link in vitro-derived pre-steady state kinetic data with a quantitative and time-resolved understanding of RNAi on the cellular level. The observation that product release by Argonaute 2 is accelerated in the presence of an excess of target RNA in vitro inspired us to suggest an associative mechanism for the RNA slicer reaction where incoming target mRNAs actively promote dissociation of cleaved mRNA fragments. This novel associative model is compatible with high multiple turnover rates of RNAi-based gene silencing in living cells and accounts for target mRNA concentration-dependent enhancement of the RNAi machinery.

Computational identification of biologically functional non-hairpin GC-helices in human Argonaute mRNA.

BACKGROUND: Perfectly formed duplex elements in RNA occur within folding units, often as a part of hairpin motifs which can be reliably predicted by various RNA folding algorithms. Double helices with consecutive Watson-Crick base-pairing may also be formed between distant RNA segments thereby facilitating long-range interactions of long-chain RNA that may be biologically functional. Here we addressed the potential formation of RNA duplex motifs by long-range RNA-RNA interactions of distantly located matching sequence elements of a single long-chain RNA. RESULTS: We generated a Python-based software tool that identifies consecutive RNA duplex elements at any given length and nucleotide content formed by distant sequences. The software tool, dubbed RNAslider, is built on the theoretical RNA structure prediction algorithm Mfold. Source code and sample data sets are available on demand. We found that a small ratio of human genes including the Argonaute (Ago)-like gene family encode mRNAs containing highly GC-rich non-hairpin duplex elements (GC-helix) of equal to or more than 8 base pairs in length and we provide experimental evidence for their biological significance. CONCLUSION: GC-helices are observed preferentially within the 5'-region of mRNAs in an evolutionarily conserved fashion indicating their potential biological role. This view is supported experimentally by post-transcriptional regulation of gene expression of a fusion transcript containing 5'-sequences of human mRNA(Ago2) harbouring GC-helices and down-stream coding sequences of Renilla luciferase.