RNA polymerase II expression vectors for RNA interference based on microRNA-155.

Abstract

The complexity and dynamics of the developing nervous system challenges the traditional approaches of reverse genetics. Vector-driven RNA interference (RNAi) provides a viable alternative as a convenient, economic and versatile research tool to inhibit gene expression. RNAi is a type of post-transcriptional gene silencing mechanism, in which small RNAs of ∼19-23 nt bind to the target mRNA sequences with perfect or near-perfect complementarity and induce cleavage, leading to sequence specific degradation of mRNAs. A novel class of endogenous small RNAs called microRNAs (miRNAs) was recently discovered to be an important regulator of gene expression at the post-transcriptional level. Varying in size between 19 and 25 nucleotides, miRNAs either inhibit the translation of partially complementary mRNAs without affecting mRNA transcript abundance, or employ an RNAi mechanism to degrade target mRNAs with a higher degree of complementarity. To trigger highly efficient RNAi by utilizing the cellular machinery for biogenesis of natural miRNAs, we have developed new RNA polymerase II expression vectors for RNAi designated synthetic inhibitory BIC-derived RNA (SIBR) vectors. The SIBR vectors use a modified miR-155 microRNA (miRNA) precursor stem-loop and flanking sequences as a cassette to express synthetic miRNAs complementary to target RNAs. By placing the SIBR cassettes within an intron of a protein-coding gene, we can coexpress a marker protein with miRNAs in a tightly correlated temporal and spatial pattern. Furthermore, multimerization of SIBR cassettes in tandem enables us either to inhibit a protein more effectively, or inhibit multiple proteins simultaneously. We utilized the unique combination of features in SIBR vectors to study the biological function of the scaffold protein POSH (Plenty of SH3 domains) during neuronal differentiation. By inhibiting POSH protein in differentiating mouse primary cortical neurons, combined with time-lapse image analysis, we demonstrate that POSH down-regulates the extension phase of axon outgrowth in cortical progenitors, without affecting neuronal survival or apoptosis. In similar experiments, the F-actin binding protein Shroom, a newly identified interacting partner of POSH, also inhibits process elongation, presumably by regulating the neuronal cytoskeleton via its actin binding domain. Combined, these results suggest that POSH and Shroom work together to negatively regulate axon outgrowth in differentiating neurons, potentially through modulation of the neuronal cytoskeleton.