Abstract 16472: The RNA-Binding Protein Quaking Regulates Alternative Splicing Events in Myocardin That Determine Vascular Smooth Muscle Cell Phenotype
Eukaryotic cells express more than 500 RNA-binding proteins (RBPs), which possess a remarkable capacity to specifically recognize (pre-)mRNA targets on the basis of sequence and/or structure. RBPs are critical regulators of post-transcriptional RNAs and can influence pre-mRNA splicing, mRNA localization and stability.
We identified that the RNA-binding protein Quaking (QKI) is highly expressed by neointimal vascular smooth muscle cells (VSMCs) of human coronary restenotic lesions, but not in healthy coronary vessels. Using a vascular injury model, mice hypomorphic for QKI (Qkv) were observed to have reduced neointimal hyperplasia as compared to wild-type mice (Qkv 1,779 ± 758 μm2 vs. WT 3,537 ± 1,147 μm2; n=6, * p<0.05). Concordantly, abrogation of QKI using shRNA (sh-qkI) attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, and conferred non-contractile VSMCs with the capacity to functionally contract on deformable substrates (sh-cont 0 ± 0% vs. sh-qkI 77 ± 12%, n=10, ***p<0.001). QKI localized to the spliceosome, where it interacts with the myocardin pre-mRNA at the alternative exon 2a, enabling QKI to directly impact the myocd_v3 and myocd_v1 mRNA transcript balance. Mutation of the QKI response element at exon 2a almost exclusively led to exon 2a inclusion, and myocd_v3 transcript formation. Furthermore, as compared to VSMCs overexpressing Myocd_v1, overexpression of Myocd_v3 in non-contractile VSMCs revealed a shift to a more contractile phenotype in combination with enhanced functional contractility. Finally, wire injury of the carotid artery in ApoE-/- mice was coupled with a striking shift in expression of myocd_v3 to myocd_v1 expression (day 0 myocd_v3: myocd_v1 95 ± 4%: 5 ± 2%; day 7 myocd_v3: myocd_v1 50 ± 12%: 50 ± 10; p<0.05, n=4 ).
Collectively, our findings suggest that QKI-induced alternative splicing of myocd in VSMCs results in the production of a Myocardin isoform that is involved in artery wall pathophysiology. As such, we propose that intervention in QKI activity could be an effective modality in the prevention of fibroproliferative disorders and the improvement of plaque stability.
- © 2013 by American Heart Association, Inc.