System Dynamics Of MiRNA Biogenesis And Transport Pathways

MicroRNAs (miRNAs) are endogenous single-stranded RNAs (ssRNAs) of~19-25-nt inlength that play a key role as post-transcriptional regulators in eukaryotes by targetingmRNAs for cleavage and degradation. They play important roles in cell growth,differentiation, proliferation, and apotosis and have been implicated in controling the timingof cell cycle exit and terminal differentiation. Therefore, deep understanding of the biogenesisand nuclear transport mechanism of miRNA will provide further insights into the importanceof miRNAs that play multiple roles in controlling both physiological and pathologicalprocesses such as the cell proliferation and cancers. Thus we characterized the dynamicproperties of miRNA biogenesis pathway on both system and molecular levels. The mainfindings of this work are summarized as follows:(1)A system-theoretic approach was presented to analyze and quantitative modeling of ageneric miRNA pathway, which can be implemented deterministically and stochastically. Ourresults show that the inferred dynamic properties obtained from the mathematical models ofthe miRNA pathway are well consistent with previous experimental observations. Bysensitivity analysis, the key steps in this pathway were found to be the miRNA genetranscription, RISC decay and mRNA formation. In addition, the results of quantified noisestrength along the pathway demonstrate that the pathway can reduce the ingress noise andreveal the noise robustness property.(2) On molecular level, we qualitatively and quantitatively analyzed the thermodynamicand kinetic mechanisms for pre-miRNA binding and export by Exp5pathway throughmolecular dynamic (MD) simulations. The results show that (a) Free Exp5undergoesextensive opening motion, and in this way facilitates the RanGTP binding.(b) RanGTPefficiently regulates the association/dissociation of pre-miRNA to its complex by inducingconformational changes in the HEAT-repeat helix stacking of Exp5.(c) The GTP hydrolysisprevents Ran from rebinding to Exp5by regulating the hydrophobic interfaces and saltbridges between Ran and Exp5.(d) The transition from the A’-form to the A-form of thepre-miRNA modulates the structural complementarities between the protein and the pre-miRNA, thus promoting efficient assembly of the complex.(e) The base-flipping process(from the closed to the fully flipped state) of the2-nt3’ overhang is a prerequisite for thepre-miRNA recognition by Exp5, which occurs in a sequence-independent manner asevidenced by the fact that different2-nt3’ overhangs bind to Exp5in essentially the sameway.(3) To fully understand the dynamic mechanism for shuttling biomolecules includingpre-miRNA between the nucleus and the cytoplasm, we examined the dynamic mechanism ofTransportin1-mediated nuclear import pathway by MD. The results reveal that (a) astrikingly intrinsic flexibility and conformational heterogeneity of Trn1, identified asgenerally segmental architecture. The segments rotate relative to each other about a flexiblehinge and thereby force Trn1to adopt a conformation compatible with the binding of Ran orsubstrates.(b) Such binding significantly suppresses the flexibility and conformationalheterogeneity of Trn1and results in a disorder-to-order transition of HR8loop, whichfacilitates this loop to allosterically communicate with the C-terminal arch of Trn1.(c)Although M9interacts strongly with both sites A and B, the high flexibility of N-terminus ofM9and HR19–HR20of Trn1would facilitate for the release of M9at site B, which controlsoverall binding affinity for Trn1.(d) The conformational changes of switches I and II of Raninduced by GTP hydrolysis directly influence and control Trn1-Ran contacts, explaining whyTrn1preferentially binds to RanGTP rather than to RanGDP.Overall, this work systemetically analyzed the dynamic properties of miRNA biogenesispathway, particularly for the regulation of the nuclear transport cycle and its underlyingmolecular interaction, which will provide full insights into miRNA biogenesis pathway.