Abstract
BackgroundRNA interference (RNAi) is a regulatory cellular process that controls post-transcriptional gene silencing. During RNAi double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous mRNA via the generation of smaller dsRNA oligomers of length between 21-23nt (siRNAs). siRNAs are then loaded onto the RNA-Induced Silencing multiprotein Complex (RISC), which uses the siRNA antisense strand to specifically recognize mRNA species which exhibit a complementary sequence. Once the siRNA loaded-RISC binds the target mRNA, the mRNA is cleaved and degraded, and the siRNA loaded-RISC can degrade additional mRNA molecules. Despite the widespread use of siRNAs for gene silencing, and the importance of dosage for its efficiency and to avoid off target effects, none of the numerous mathematical models proposed in literature was validated to quantitatively capture the effects of RNAi on the target mRNA degradation for different concentrations of siRNAs. Here, we address this pressing open problem performing in vitro experiments of RNAi in mammalian cells and testing and comparing different mathematical models fitting experimental data to in-silico generated data. We performed in vitro experiments in human and hamster cell lines constitutively expressing respectively EGFP protein or tTA protein, measuring both mRNA levels, by quantitative Real-Time PCR, and protein levels, by FACS analysis, for a large range of concentrations of siRNA oligomers.ResultsWe tested and validated four different mathematical models of RNA interference by quantitatively fitting models' parameters to best capture the in vitro experimental data. We show that a simple Hill kinetic model is the most efficient way to model RNA interference. Our experimental and modeling findings clearly show that the RNAi-mediated degradation of mRNA is subject to saturation effects.ConclusionsOur model has a simple mathematical form, amenable to analytical investigations and a small set of parameters with an intuitive physical meaning, that makes it a unique and reliable mathematical tool. The findings here presented will be a useful instrument for better understanding RNAi biology and as modelling tool in Systems and Synthetic Biology.
Highlights
RNA interference (RNAi) is a regulatory cellular process that controls post-postranscriptional gene silencing
In order to model the effects of RNA interference on mRNA expression levels at different concentration of small interfering RNA oligomers (siRNAs) oligomers, we carried out in-inivo experiments of RNA interference on two mammalian cell-cellines stably expressing the EGFP protein or the tTA protein, respectively
In the first set of experiments, Human Embryonic Kidney cells stably expressing EGFP (HEK293-EGFP cell-celline), were transfected with varying quantities of synthetic siRNA oligomers directed against the EGFP mRNA in the range of 0 to 200 pmol
Summary
RNA interference (RNAi) is a regulatory cellular process that controls post-postranscriptional gene silencing. During RNAi double-doubletranded RNA (dsRNA) induces sequence-sequencepecific degradation of homologous mRNA via the generation of smaller dsRNA oligomers of length between 21-23nt (siRNAs). SiRNAs are loaded onto the RNA-Induced Silencing multiprotein Complex (RISC), which uses the siRNA antisense strand to recognize mRNA species which exhibit a complementary sequence. RNA interference (RNAi) is a well characterized regulatory mechanism in eukaryotes [1,2,3] as well as a powerful tool for understanding gene function, thanks to the discovery that synthetic small interfering RNA oligomers (siRNAs) can efficiently induce RNAi in mammalian cells [4,5]. In step 1, the presence of double stranded RNA (dsRNA) elicits a response in the cell mediated by the Dicer enzyme, which binds and cleaves the dsRNA into fragments of 21-23 base pairs, called small interfering RNA (siRNA). In step 4, mRNA degradation is induced, target mRNA is dissociated from the siRNA, and the mRNA EGFP (%)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have