Irip alternative4/2/2023 ![]() Total RNA was extracted from HK-2 cells with TRIzol Reagent (Life Technologies Corporation) and quantified using a NanoDrop™ 3000 Spectrophotometer (Thermo Fisher Scientific) according to the manufacturer's instructions. After 48 h, cells were collected for follow-up experiments. shPARP1 plasmids (GenePharma, Suzhou, China) (2.5 μg) were transfected into HK-2 cells using Lipofectamine 2000 Reagent (Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer's instructions. For knockdown of PARP1, HK-2 cells were incubated until cells were 60–80% confluent. Human renal tubular epithelial cell line HK-2 was cultivated in Dulbecco's modified Eagle's medium/F12 (H圜lone, Rockville, MD, USA) containing 10% FBS, penicillin (10 units per μL), and streptomycin (10 units per mL) (Gibco, Carlsbad, CA, USA) at 37 ☌ in an incubator (Thermo Fisher Scientific, Marietta, OH, USA). Materials and methods Cell culture and transfection Our work defined a complex genome-wide PARP1–RNA interaction map in a human proximal tubular epithelial cell line and furthered our understanding of the regulatory role of PARP1 at the pre-mRNA level in HK-2 cells. In the present study, we have explored the mechanism by which PARP1/RNA interactions regulated gene expression in HK-2 cells by obtaining PARP1-regulated transcriptomes using RNA-seq and iRIP (improved RNA immunoprecipitation)-seq. In the latter, doxorubicin-induced DNA damage and cytosolic ROS have been found to be mediated by PARP1 via independent pathways ], which prompted us to further investigate PARP1 targets and their regulatory role in HK-2 cells. In addition, PARP1 hyperactivity and elevation of intracellular Ca 2+ are responsible for the amplification of ROS-induced nonapoptotic cell death in HK-2 proximal tubular epithelial cells ]. In these, the renal disease presents itself with various degrees of renal proximal tubular epithelial cell injury. Moreover, PARP1 is associated with several types of renal diseases, such as renal failure ], renal cell carcinoma ], and renal ischemia–reperfusion injury ]. PARP1 was found to preferentially bind RNA-containing GC-rich sequences, and 20 484 sites were located in intron regions ]. ![]() Recently, a CLIP-seq study of PARP1 RNA targets in human HeLa cells identified an overlapping set of 22 142 PARP1 RNA-binding peaks mapped to mRNAs. PARP1 participates in regulation of the gene expression at both transcription initiation and pre-mRNA splicing levels by binding to RNA, splicing factors, and chromatin ]. Poly(ADP-ribose) polymerase 1 (PARP1) has been reported to be involved in RNA synthesis and transcription ], mRNA stability ], and variable splicing of RNA ]. ![]() RBPs bind to these target sequences forming ribonucleoprotein complexes ], which regulate transcription, pre-mRNA processing, and translation ]. To understand the regulatory roles of RBPs in gene expression, their RNA target sites must be identified. ![]() RNA-binding proteins (RBPs) are involved in the regulation of both physiological and pathological states ]. STRING, Search Tool for the retrieval of interacting genes RASGs, regulated alternative splicing genes ![]()
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