SAT0065 Chondrocytes under pathological conditions preferentially degrades selected trnas to inhibit protein synthesis
Authors: J. Green, T. Haqqi
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Oxidative and ER stress play an important role in the pathogenesis of osteoarthritis (OA). These stress conditions can induce the cleavage of transfer RNA (tRNA) via angiogenin. This cleavage at the anticodon loop of the tRNA produces 5'-tRNA and 3'-tRNA halves known as tiRNAs. <h3>Objectives</h3> We aim to identify specific tRNA to tiRNA isotype fragments that occur during oxidative or ER stress in human OA chondrocytes. <h3>Methods</h3> Primary human articular chondrocytes were enzymatically isolated from OA cartilage. Cells were cultured <i>in vitro</i> for 72 hour and then treated with tert-butyl hydroperoxide (TBHP) over a 12 hour time course or with Interleukin-1b (IL1b) over a 24 hour time course. RNA was collected for mRNA expression analysis (qRT-PCR). Protein was harvested for western blot analysis. A tRF and tiRNA PCR Array that profiles 55 known tiRNAs was used to perform an unbiased dissection of the formation of tiRNAs in OA chondrocytes. Total RNA was adapted using a First-Strand cDNA Synthesis Kit designed to create cDNA libraries from small RNAs for qPCR detection to identify ten novel tiRNAs associated with cell stress in OA chondrocytes. <h3>Results</h3> Using qRT-PCR RNase gene expression was measured. Of these RNases 250 µM TBHP treatment at 12 hour induced KIAA0430 (p&lt;0.05), RNase7 (p&lt;0.0001) and ZC3H12A (p&lt;0.0001) but not angiogenin or SND1. 10 ng/ml IL1ß treatment at 6 hour induced SND1 (p&lt;0.0001), RNase 7 (p&lt;0.01), ZC3H12A (p&lt;0.01) but not angiogenin or KIAA0430. Chondrocytes undergoing oxidative or ER stress produced tiRNA halves in a time dependent manner, tiRNA formation was highest at 6 hour of IL1ß treatment and after 12 hour of TBHP treatment. tiRNA fragments of tRNAs specific for arginine, glutamic acid, glycine, histidine, lysine and valine were increased after TBHP or IL1ß treatment. Ten tiRNAs that were induced by IL1ß and TBHP were selected for further study. Primer sets for these tiRNA and the parent tRNAs were designed and screened across a wider time course, and with higher patient numbers. <h3>Conclusions</h3> The expression of several RNases was increased in a time dependent manner in stressed OA chondrocytes and may be involved in the cleaveage of tiRNA fragments. tiRNA halves were grouped together based on their parent tRNA amino acid target. We hypothesised that an increase in fold change in these grouped tiRNAs may result in less production of proteins that contain a high percentage of that specific amino acid. These changes in amino acid distribution may account for decreases seen in amino acid rich proteins such as Type II collagen (a glycine rich protein) in stressed chondrocytes. By designing tiRNA and parent tRNA primers we will discover if tiRNA formation effects total levels of parent tRNAs. The outcomes from this research will provide us with an increased understanding of tRNA to tiRNA formation in human chondrocytes and may allow us to identify therapeutic targets for the treatment of OA and provide insights into novel stress signalling pathways. <h3>Acknowledgements</h3> This work was supported by funds from the Northeast Ohio Medical University to TMH. <h3>Disclosure of Interest</h3> None declared