A reader of this blog asked about the impact on our understanding of microRNA biology of a paper that was just published in Cell and shows microRNA-328 to function both as a traditional RNA silencing agent and molecular decoy for an RNA binding protein (Eiring et al. [2010]. miR-328 functions as an RNA decoy to modulate hnRNP E2 regulation of mRNA translation in leukemic blasts). The paper demonstrates that miR-328 binds tightly to an RNA binding protein, hnRNP E2, so that it is no more available to act as a suppressor of C/EBPalpha translation through the recognition of a miR328-related sequence element in the C/EBPalpha mRNA. This is a critical event for triggering the blast crisis stage of chronic myelogenous leukemia. The finding that a microRNA can act as a competitive inhibitor of sequence-specific regulators of RNA processing, in addition to its well known function in RNA silencing, not only necessitates a re-evaluation of the biological network that microRNAs operate in, but raises the prospect for new (small) RNA Therapeutics approaches.
Discovery of miR-328 as a competitor for hnRNP E2 binding
HnRNP E2, a protein that belongs to a class of proteins originally described to be highly abundan proteins in the nucleus that bind to RNA, has been known repress the translation of C/EBPalpha by binding to a C-rich element of that mRNA. Since C/EBPalpha is critical for myeloid differentiation of blood stem cells, hnRNP E2 ensures that a proper ratio of precursor to differentiated cells is maintained. A remaining puzzle in the understanding of this regulation, however, was the observation that myeloid precursor cell proliferation could still occur in certain situations when hnRNP E2 levels are high without evidence for a post-translational modification of hnRNP E2 that might account for its loss of C/EBPalpha mRNA-repression activity.
Since small non-coding RNAs, particularly microRNAs, are implicated in cell proliferation and differentiation as well as in related disease such as cancer, the authors of the paper hypothesized that it is a microRNA that negatively modulated hnRNP E2-mediated translation repression activity by competitively binding to it, and that the loss of such regulation is responsible for the differentiation deficit in the blast crisis stage of chronic myelogenous leukemia (as opposed to the more indolent chronic phase of the disease). I have to admit that postulating a microRNA decoy activity would have seemed like a risky move at this point, but by following through with microRNA expression profiling, they indeed were able to come up with a microRNA, miR-328, that fulfilled the criteria for such an activity: its diminished abundance/loss during blast crisis CML and a C-rich element akin to the one found in C/EBPalpha mRNA.
Sure enough, a battery of binding experiments demonstrated that miR-328 was an efficient competitor for hnRNP E2 binding and that this freed C/EBPalpha mRNA which was associated with its increased translation into protein and myeloid differentiation. The reason why miR-328 was essentially lost in (blast crisis) CML was due to over-active signaling downstream of the BCR-ABL mutant protein which is characteristic for CML.
Therapeutic RBP Decoys?
The finding that a microRNA may act as a sequence-specific molecular decoy for an RNA-binding protein raises the possibility that microRNA decoy mimicry or inhibition strategies could be therapeutic. While much more work remains to be done to validate miR-328 as a good candidate for microRNA decoy mimicry in CML by restoring myeloid differentation, it will be important to find out whether other microRNAs live similar double-lives. Though not trivial, a careful analysis of the direct interactions of microRNAs with other RNA binding proteins (RBPs) might yield additional candidates. Such therapeutic approaches would not even have to be limited to microRNAs but could encompass all kinds of natural and synthetic RNA-binding protein decoys. In fact, an RNA decoy mimicking the TAR element of HIV RNA is part of the triple (shRNA, ribozyme, decoy)-agent developed by the City of Hope and Benitec, and DNA transcription factor decoy approaches have also been conceived. Of course, such therapeutic approaches could piggy-back on many of the advances already made for other RNA therapeutic modalities before, including delivery and nucleic acid chemistries. This also means various start-up and business development opportunities for existing companies (e.g. out-licensing of established delivery technologies).
Beyond that, however, I would caution that such decoys should be much harder to develop as a platform since each RBP-RNA relationship would have to be looked at in detail in order to understand for example which exact sequence and structural motif is involved and where in the cell one has to intervene. In addition, although some would argue that it is actually a potential advantage, since most RBPs bind to multiple RNAs, a therapeutic approach has to take into account the complex systems effect that it could trigger, somewhat reminiscent of microRNA Therapeutics. By contrast, once the genetic relationship is understood, the goal for RNAi Therapeutics is always the same: get the RNAi trigger into the cytoplasm of the target cell to surgically remove one gene at a time. It is this basic simplicity that has always been at the bottom of my fascination for RNAi Therapeutics.
Great review of the paper!
ReplyDelete