SPRTN-dependent DPC degradation precedes repair of damaged DNA: a proof of concept revealed by the STAR assay.

DNA-protein crosslinks (DPCs), formed by the covalent conjugation of proteins to DNA, are toxic lesions that interfere with DNA metabolic processing and transcription. The development of an accurate biochemical assay for DPC isolation is a priority for the mechanistic understanding of their repair. Here, we propose the STAR assay for the direct quantification of DPCs, sensitive to physiologically relevant treatment conditions. Implementing the STAR assay revealed the formation of small cross-linked peptides on DNA, created by the proteolytic degradation of DPCs by SPRTN. The initial proteolytic degradation of DPCs is required for the downstream activation of DNA repair, which is mediated through the phosphorylation of H2Ax. This leads to the accumulation of DNA repair factors on chromatin and the subsequent complete removal of the cross-linked peptides. These results confirmed that the repair of DPCs is a two-step process, starting with proteolytic resection by SPRTN, followed by the repair of the underlying damage to the DNA.

Direct and cost-effective method for histone isolation from cultured mammalian cells.

Histones are an essential part of nucleosomes that regulate chromatin structure and function. Histone exchanges and modifications represent a scaffold for DNA transcription, repair, and replication. Studying histones and histone code is an important and fast-developing branch of epigenetic science. Here we propose a fast, efficient, and versatile assay for nucleosomal histone isolation from mammalian cells, without the use of acids or high salt solutions which are common for other histone isolation techniques. All components used in the protocol are common and inexpensive laboratory chemicals. The protocol has been evaluated on six commonly used cell lines and two animal tissue samples. The mild extraction conditions preserve delicate histone epigenetic changes, allowing its downstream analyses. We have demonstrated the assays' successful application during changes in the transcriptional activity of histone genes, cell cycle transitions, and DNA-damaging conditions. Histone fractions, obtained by the protocol, can be used for further applications, such as electrophoresis, immunoblot, and mass spectrometry. Therefore, the new proposed nucleosomal histone isolation method is sensitive, specific, and suitable for downstream applications of various kinds.

Aberrations in FGFR1, FGFR2, and RIP5 Expression in Human Congenital Anomalies of the Kidney and Urinary Tract (CAKUT).

This study aimed to explore the spatio-temporal expression patterns of congenital anomalies of kidney and urinary tract (CAKUT) candidate genes, Fibroblast Growth Factor Receptor 1 (FGFR1), Fibroblast Growth Factor Receptor 2 (FGFR2) and Receptor-Interacting Protein Kinase 5 (RIP5), in human fetal kidney development (CTRL) and kidneys affected with CAKUT. Human fetal kidneys from the 22nd to 41st developmental week (duplex, hypoplastic, dysplastic, and controls) were stained with antibodies and analyzed by epifluorescence microscopy and RT-qPCR. The effect of CAKUT candidate genes on kidney nephrogenesis and function is confirmed by statistically significant variations in the spatio-temporal expression patterns of the investigated markers. The nuclear localization of FGFR1, elevated expression score of FGFR1 mRNA, the increased area percentage of FGFR1-positive cells in the kidney cortex, and the overall decrease in the expression after the peak at the 27th developmental week in dysplastic kidneys (DYS), suggest an altered expression pattern and protein function in response to CAKUT pathophysiology. The RT-qPCR analysis revealed a significantly higher FGFR2 mRNA expression score in the CAKUT kidneys compared to the CTRL. This increase could be due to the repair mechanism involving the downstream mediator, Extracellular Signal-Regulated Kinase 1/2 (ERK1/2). The expression of RIP5 during normal human kidney development was reduced temporarily, due to urine production and increased later since it undertakes additional functions in the maturation of the postnatal kidney and homeostasis, while the expression dynamics in CAKUT-affected kidneys exhibited a decrease in the percentage of RIP5-positive cells during the investigated developmental period. Our findings highlight the importance of FGFR1, FGFR2, and RIP5 as markers in normal and pathological kidney development.

Identification of a novel lncRNA induced by the nephrotoxin ochratoxin A and expressed in human renal tumor tissue.

Long non-coding RNAs represent a fraction of the transcriptome that is being increasingly recognized. For most of them no function has been allocated so far. Here, we describe the nature and function of a novel non-protein-coding transcript, named WISP1-AS1, discovered in human renal proximal tubule cells exposed to the carcinogenic nephrotoxin ochratoxin A. WISP1-AS1 overlaps parts of the fourth intron and fifth exon of the Wnt1-inducible signaling pathway protein 1 (WISP1) gene. The transcript is 2922 nucleotides long, transcribed in antisense direction and predominantly localized in the nucleus. WISP1-AS1 is expressed in all 20 samples of a human tissue RNA panel with the highest expression levels detected in uterus, kidney and adrenal gland. Its expression was confirmed in primary tissues of human kidneys. In addition, WISP1-AS1 is expressed at higher levels in renal cell carcinoma (RCC) cell lines compared to primary proximal tubule cells as well as in RCC lesions than in the adjacent healthy control tissue from the same patient. Using specific gapmer antisense oligonucleotides to prevent its upregulation, we show that WISP1-AS1 (1) does not influence the mRNA expression of WISP1, (2) affects transcriptional regulation by Egr-1 and E2F as revealed by RNA-sequencing, enrichment analysis and reporter assays, and (3) modulates the apoptosis-necrosis balance. In summary, WISP1-AS1 is a novel lncRNA with modulatory transcriptional function and the potential to alter the cellular phenotype in situations of stress or oncogenic transformation. However, its precise mode of action and impact on cellular functions require further investigations.

SPRTN is involved in hepatocellular carcinoma development through the ER stress response.

Endoplasmic reticulum (ER) stress, prompted by the accumulation of misfolded or unfolded proteins, triggers the activation of the unfolded protein response (UPR) pathway to restore ER homeostasis. This stress response is implicated in the development of hepatocellular carcinoma (HCC). A biallelic mutation in SPRTN is currently the only known single-gene mutation implicated in the early onset of HCC. However, the exact mechanism linking SPRTN mutations to HCC remains unclear. In our study, we analyzed SPRTN and UPR in 21 human HCC tissue samples using RT-qPCR, immunoblot, and immunohistochemistry. We found alterations in the expression levels of SPRTN and UPR-related genes and proteins in HCC samples. The impact of SPRTN on the ER stress response was assessed in SPRTN-depleted HepG2 cells through RNA sequencing, pull-down assay, comet assay, and mitotic index calculation. We demonstrated that SPRTN interacts with the UPR sensor GRP78. Furthermore, we observed a decrease in SPRTN levels during ER stress, and increased sensitivity to ER stress in SPRTN-depleted cells. These findings suggest an essential role for SPRTN in the ER stress response and provide new insights into HCC pathogenesis. This newly discovered function of SPRTN could significantly enhance our understanding and treatment of HCC.