Downregulation of hnRNPK mimics the “Abi-1 depletion phenotype” in neurons.

<p>(A) hnRNPK-RNAi knockdown in NIH3T3 cells with a construct targeting the 3′UTR-region of hnRNPK. (I) hnRNPK is endogenously expressed in NIH3T3, HeLA and Cos7 cells and is detectable in cultured hippocampal neurons (DIV21). (II) After transfection of NIH3T3 cells with an hnRNPK-RNAi construct for 3 days, the cells were fixed and stained with an antibody against hnRNPK. Only untransfected cells in close proximity to the RNAi-transfected cell in the upper right are immunopositive for hnRNPK, with a predominant distribution of the protein in the nucleus. (III) After transfection of NIH3T3 cells with an hnRNPK-RNAi construct for 3 days, protein expression of hnRNPK is markedly suppressed as confirmed by Western blotting compared to vector control transfected cells. (IV) Double transfections using two different hnRNPK-RNAi constructs, one targeting the 3′UTR of the hnRNPK sequence and one targeting the coding sequence of hnRNPK together with an hnRNPK-Myc-construct which is resistant against RNAi due to 4 nucleotide exchanges in the RNA leading to an unaltered amino acid sequence. The staining against the Myc-tag shows a decreased protein level solely when using the non-resistant construct together with the RNAi targeting the CDS (coding sequence). (B) Neuronal transfection of Abi-1-RNAi and hnRNPK-RNAi constructs. (I) In contrast to the control vector (pSuper), transfection of RNAi constructs resulted in an obvious change of neuronal morphology. The downregulation of hnRNPK as well as Abi-1 is leading to an extended and extremely branched dendritic tree. (II) The number of branching points within the dendritic compartment is significantly upregulated in both RNAi groups compared to the control transfection. (III) The analysis of the dendritic tree shows a significant shift of dendrites towards small, filopodia-like tertiary dendrites. Scale bars are as indicated.