University of Heidelberg

RNAi - Background

  • Discovered 1998 in C. elegans and soon after validated in mammals including humans, the naturally occurring process of RNAi has quickly taken center stage as an exciting novel endogenous mechanism of gene regulation, and concurrently as one of our most promising options for therapeutic intervention with human diseases with an underlying genetic cause. In human cells, RNAi typically starts with the expression of a pri(mary) mi(cro)RNA in the nucleus which is then trimmed to a pre(cursor) miRNA and subsequently exported into the cytoplasm by Exportin-5. There, it is further cropped to the mature miRNA which is finally loaded into an RNA-induced silencing complex (RISC). The latter then binds and suppresses a target mRNA that is partially or fully complementary to the miRNA.
  • A major component of RISC are the Argonaute (Ago) proteins which in humans constitute a family of four proteins, Ago1 through 4. All consist of four distinct domains labeled N, MID, PAZ and PIWI that act together in loading the mature miRNA and in unwinding the double-stranded RNA to yield a single-stranded molecule which can then bind the target mRNA. Curiously, the exact function of, and difference between, the four human Ago proteins remains ill understood to date. One notable exception is Ago2 which is also called Slicer due to its unique ability to cleave a bound target mRNA at a precise location, resulting in more potent gene suppression than the mRNA destabilization induced by Ago1, 3 or 4 and the accessory proteins which they recruit (including deadenylases and the decapping machinery).
  • Today, we are aware of >1500 miRNAs that regulate gene expression in human cells and that are critically involved in all essential cellular processes, from cell division or growth to senescence and apoptosis. Unsurprisingly, evidence has likewise accumulated that miRNAs or the RNAi machinery in general are crucial players in human pathogenesis. Outstanding examples are cancers and viral infections which are frequently characterized by marked dysregulations of RNAi components, such as down-regulation or inhibition of the core protein factors. Very typical in particular for cancers is also a suppression of numerous miRNAs, whereas viral infections often invoke both miRNA up- and down-regulations. The underlying molecular mechanisms remain largely unclear but may affect any step in the miRNA biogenesis pathway.
  • Intriguingly, a wealth of very recent studies demonstrate that miRNAs are not only present within cells but are also detectable in extra-cellular environments, including blood (serum or plasma), saliva, urine or tear fluid. The mechanisms by which these miRNAs are secreted from cells (as naked RNAs, in exosomes or complexed with proteins), whether this release is an active process or rather a by-product of cell death and lysis, and whether and how circulating blood-borne miRNAs are taken up by new cells in order to regulate their gene expression, are all intriguing questions at this point. Already clear is that the profiles of these extra-cellular miRNAs can be associated with human pathologies, most notably cancer and viral infections, implying their high usefulness as easily accessible new and specific biomarkers of disease.
  • Importantly, RNAi is not only an endogenous mechanism, but can also be deliberately induced exogenously via artificial triggers that mimic cellular RNAi molecules. As shown in the upper half, these can be shmiRNAs which mimic pri-miRNAs, short hairpin or shRNAs which resemble pre-miRNAs, or small interfering or siRNAs which are the equivalent of mature miRNAs (in their double-stranded form). With the exception of siRNAs, these artificial RNAi inducers can be expressed from RNA polymerase II or III promoters and are thus inherently compatible with vector-based gene delivery or therapy strategies. However, careful dosing of intra-cellular shRNA expression levels is critical as evidenced by our previous reports that saturation of the cellular RNAi machinery causes cytotoxicity (Grimm et al., Nature 2006 & J. Clin. Invest. 2010).
  • Yet another powerful option to exploit RNAi for therapeutic purposes is to correct aberrant miRNA expression in diseased cells, i.e. to either over-express miRNAs that are down-regulated e.g. in cancers, or to inhibit miRNAs that are up-regulated e.g. as a consequence of viral infections. This strategy is particularly potent and promising in combination with viral gene transfer vectors such as those that we develop in our lab based on AAV, which can efficiently and specifically deliver cassettes for miRNA regulation to desired cells. A most remarkable potential target for such a miRNA-based therapy is miR-122, a liver-specific miRNA that is critically needed by HCV for its replication. In fact, to be able to ultimately block this miRNA in vivo with tailored AAV vectors is one of our essential goals in the lab.
Contact: E-Mail (Last update: 27/02/2012)