Project description:Emerging studies have revealed diverse cellular functions of tRNA-derived small RNAs (tsRNAs, or tDRs). Here, we show that a hypoxia-induced tDR, derived from the 3’ end of tRNA-Asp-GTC (tRNA-Asp-GTC-3’tDR), activates, while its inhibition blocks autophagic flux in kidney cells. Functional gain/loss-of-function studies in murine kidney disease models demonstrate a significant reno-protective function of tRNA-Asp-GTC-3’tDR. Mechanistically, tRNA-Asp-GTC-3’tDR assembles stable G-quadruplex structures and sequesters pseudouridine synthase PUS7, preventing catalytic pseudouridylation of histone mRNAs. The resulting pseudouridylation deficiency directs histone mRNAs to the autophagosome-lysosome pathway, triggering RNA autophagy. We confirm this tDR-induced RNA autophagy pathway in murine and human kidney diseases. Together, our findings identify a novel role for tRNA-Asp-GTC-3’tDR in regulating RNA autophagy in kidney cells to maintain homeostasis and protect against kidney injury.
Project description:Long interspersed element 1 (LINE-1, or L1) is a retrotransposon that constitutes ~17% of the human genome. Although ~6,000 full-length L1s spread throughout the human genome, their biological significance remains undetermined. L1 5’ untranslated region has a bidirectional promoter activity with a sense promoter, driving L1 mRNA, and an antisense promoter (ASP), driving L1-gene chimeric RNAs. Here, we stimulated L1 ASP activity using the CRISPR-Cas9 system to evaluate its biological impacts. Activation of L1 ASP enhances L1 retrotransposition and cell growth. Conversely, we identified epigallocatechin gallate (EGCG) as an inhibitor of L1 ASP. The inhibition of L1 ASP by EGCG decreased cell growth but did not affect L1 retrotransposition. Collectively, these results indicate that activation of L1 ASP activity fuels cell growth. To our knowledge, this is the first report demonstrating the role of L1 ASP in the biological process. Considering that L1 sequences are de-silenced in various tumor cells, activation of L1 ASP may be a cause of tumor growth and interfering with L1 ASP activity will be a potential strategy to suppress the growth.
Project description:Single cell RNA sequencing (scRNA-seq) analysis identified notochordal nucleus pulposus (NP) cells and chondrocyte-like NP cells in NP. Cells in human induced pluripotent stem cell-derived cartilage (hiPS-Cart) corresponded to chondrocyte-like NP cells but not to notochordal NP cells. hiPS-Cart cells changed their profile after implantation into intervertebral disks of nude rats, differentiating into two lineages that are metabolically distinct from each other. However, post-implanted hiPS-Cart cells corresponded to chondrocyte-like NP cells only and did not develop into notochordal NP cells.
Project description:P. aeruginosa possesses the ability to utilize a wild range of compounds as the sole source of carbon and nitrogen, including proteogenic amino acids. In particular, utilization of L-Asp and L-Asn is insensitive to carbon catabolite repression as strong growth retains in the cbrAB mutants devoid of the essential regulators for the activation of most catabolic genes. Transcriptome analysis and functional characterization were conducted to identify genes that participate in the catabolism, uptake, and regulation of these two amino acids. Through gene knockout and growth phenotype analysis, degradation of L-Asn to L-Asp was shown to be mediated by two asparaginases AsnA and AsnB, whereas only AnsB is required for the deamidation of D-Asn to D-Asp. While D-Asp is a dead-end product, conversion of L-Asp to fumurate is catalyzed by an aspartase AspA as further evidenced by enzyme kinetics. The results from the measurements of promoter-lacZ expression in vivo and mobility shift assays in vitro demonstrated that the asnR and aspR genes encode two transcriptional regulators in response to L-Asn and L-Asp, respectively, for the induction of the ansPA operon and the aspA gene. In addition, exogenous L-Glu also cause induction of the aspA gene, most likely due to its conversion to L-Asp by the aspartate transaminase AspC. Expression of several transporters were also found inducible by L-Asn and/or L-Asp, including AatJQMP for acid amino acids, DctA and DctPQM for C4-dicarboxylates, and PA5530 for C5-dicarboxylates. In summary, a complete pathway and regulation for L-Asn and L-Asp catabolism was established in this study. Cross induction of three transport systems for dicarboxylic acids may provide a physiological explanation for the insensitivity of L-Asn and L-Asp utilization to carbon catabolite repression.
Project description:We report here bulk RNA sequencing results of control and MAPK/ERK-deficient ureteric bud (UB) epithelium and nephron progenitor cells (NP) in the developing mouse kidney. RNA isolated from 4 biological replicates for UB and 3 biological replicates for NP was subjected to the library preparation done using NuGen Ovation Solo. Sequencing with NextSeq was performed at BIDGEN DNA Sequencing, after data processing produced 1004 (UB) and 5053 (NP) differentially expressed genes with a statistical cutoff of Padj<0.05 and a magnitude threshold of │log2foldchange│≥1 .
Project description:We determined the gene expression profiles of murine melan-a melanocytes treated with ASP or alpha-MSH over a 4 days time course using genome-wide oligonucleotide microarrays. As expected, the gene expression patterns emphasized the opposing effects of the 2 ligands, and there were significant reductions in expression of numerous melanogenic proteins elicited by ASP, which correlates with its inhibition of pigmentation. However, ASP also unexpectidly modulated the expression of genes involved in various other cellular pathways, including glutathione synthesis and redox metabolism. Many genes up-regulated by ASP are involved in morphogenesis, cell adhesion and ECM-receptor interactions.