Final Report Summary - RNAREGMAP (Condition specific RNA Regulatory Maps)
Objectives:
This project was designed to determine specific mRNA regulatory factors and elements important for a given biological process, in this case human steroid hormone metabolism. These regulatory factors include RNA-binding proteins (RBPs), microRNA, and long non-coding RNA (lncRNA). Specifically, the goal was to construct a transcriptome-wide post-transcriptional regulatory map of RBP-mRNA that could be used to A) identify RBPs inportant for hormone metabolism and B) the post-transcriptional regulatory for how they conrol target gene expression. Since the beginning of the project, we have made substantial progress identifying numerous candidate RBPs and even noncoding RNA. We have also investigated the phenotypic impact of a specific RBP in vitro and in vivo.
Results:
We modified our existing computational infrastructure and examined ~80 global RBP-RNA datasets representing 44 RBPs with diverse functions including pre-mRNA spicing and poly-adenylation, snoRNA biogenesis, microRNA-mediated regulation, nonsense-mediated decay, and regulators of RNA stability and translation (Figure 1).
We have optimized genomic methods to calculating RNA production, processing and decay rates in human cell lines (Figure 2A). Our data is highly reproducible and effective for calculating rates, particularly after labeling RNA for at least 15 minutes (Figure 2B). We found that lncRNAs are substantially less stable than protein-coding RNA (Figure 2C).
We have established a human adrenocortical cell culture model and performed numerous genomic assays identifying genes regulated in response to Angiotensin and Forskolin, key compounds stimulating hormone production. These assays determine the step in gene expression that is changing, e.g. transcription, splicing, stability, and translation. We recapitulate known changes in gene expression and additionally identify all putative regulatory TFs, RBPs, microRNAs, and lncRNAs (Figure 3).
We have investigated the role of the RBP ZFP36L2 in adrenal hormone metabolism in human and mouse. Many of the genes regulated upon Angiotensin treatment in the human cell line also exhibit changes in expression between the WT and KO ZFP36L2 mouse. The mouse experiments were carried out by our collaborator in the United States and we did not handle any live mice. We identified numerous responsive miRNA, including miR-21-5p that is a known regulator of aldosterone production. Interestingly, ZFP36L2 has a conserved mir-21-5p binding site in its 3’ UTR, suggesting the possibility of a negative feedback loop.
We identified translated ORFs for over 10,000 protein-coding genes and ~50 ORFs in non-coding RNA that show evidence of translation (Table 1).
Conclusions and Impact:
Our research impacts both post-transcriptional gene regulation and the mechanisms of human steroid hormone metabolism. We have identified coherent modules in RBP-RNA networks regulating specific processes such as cleavage and polyadenylation, mRNA stability, and translation. We have performed the most thorough analysis to date of metabolic labeling of RNA to calculate rates of synthesis, processing, and degradation. These results show clear technical and biological insights that will be important to the field of gene expression. Our results have expanded the known repertoire of gene expression regulators of adrenocortical steroid hormone metabolism. In this system, we have generated a comprehensive identification of regulators of RNA metabolism, specifically RBPs, miRNAs and lncRNA. Furthermore, our work focusing on ZFP36L2 identifies an important post-transcriptional regulator of steroid hormone metabolism. These results have the potential to impact adrenal diseases, specifically form of primary hyperaldosteronism. Finally, we expect to submit two manuscripts as a result of this work before the end of the calendar year.
Target groups:
Our research affects numerous target groups. Inappropriate production of hormone produced in the adrenal cortex causes major physiological disorders given the potent nature of these hormones. This includes diseases based on the overproduction of adrenal cortisol such as Cushing’s syndrome. Another major category is Conn’s syndrome and other forms of primary aldosteronism, which may impact up to 20% of hypertensive population. Due the gender differences in the presentation of primary aldosteronism, this work has the potential to more greatly benefit female health. This is important due to reported gender imbalance in animal studies and human clinical trials resulting in less female subjects.
This project was designed to determine specific mRNA regulatory factors and elements important for a given biological process, in this case human steroid hormone metabolism. These regulatory factors include RNA-binding proteins (RBPs), microRNA, and long non-coding RNA (lncRNA). Specifically, the goal was to construct a transcriptome-wide post-transcriptional regulatory map of RBP-mRNA that could be used to A) identify RBPs inportant for hormone metabolism and B) the post-transcriptional regulatory for how they conrol target gene expression. Since the beginning of the project, we have made substantial progress identifying numerous candidate RBPs and even noncoding RNA. We have also investigated the phenotypic impact of a specific RBP in vitro and in vivo.
Results:
We modified our existing computational infrastructure and examined ~80 global RBP-RNA datasets representing 44 RBPs with diverse functions including pre-mRNA spicing and poly-adenylation, snoRNA biogenesis, microRNA-mediated regulation, nonsense-mediated decay, and regulators of RNA stability and translation (Figure 1).
We have optimized genomic methods to calculating RNA production, processing and decay rates in human cell lines (Figure 2A). Our data is highly reproducible and effective for calculating rates, particularly after labeling RNA for at least 15 minutes (Figure 2B). We found that lncRNAs are substantially less stable than protein-coding RNA (Figure 2C).
We have established a human adrenocortical cell culture model and performed numerous genomic assays identifying genes regulated in response to Angiotensin and Forskolin, key compounds stimulating hormone production. These assays determine the step in gene expression that is changing, e.g. transcription, splicing, stability, and translation. We recapitulate known changes in gene expression and additionally identify all putative regulatory TFs, RBPs, microRNAs, and lncRNAs (Figure 3).
We have investigated the role of the RBP ZFP36L2 in adrenal hormone metabolism in human and mouse. Many of the genes regulated upon Angiotensin treatment in the human cell line also exhibit changes in expression between the WT and KO ZFP36L2 mouse. The mouse experiments were carried out by our collaborator in the United States and we did not handle any live mice. We identified numerous responsive miRNA, including miR-21-5p that is a known regulator of aldosterone production. Interestingly, ZFP36L2 has a conserved mir-21-5p binding site in its 3’ UTR, suggesting the possibility of a negative feedback loop.
We identified translated ORFs for over 10,000 protein-coding genes and ~50 ORFs in non-coding RNA that show evidence of translation (Table 1).
Conclusions and Impact:
Our research impacts both post-transcriptional gene regulation and the mechanisms of human steroid hormone metabolism. We have identified coherent modules in RBP-RNA networks regulating specific processes such as cleavage and polyadenylation, mRNA stability, and translation. We have performed the most thorough analysis to date of metabolic labeling of RNA to calculate rates of synthesis, processing, and degradation. These results show clear technical and biological insights that will be important to the field of gene expression. Our results have expanded the known repertoire of gene expression regulators of adrenocortical steroid hormone metabolism. In this system, we have generated a comprehensive identification of regulators of RNA metabolism, specifically RBPs, miRNAs and lncRNA. Furthermore, our work focusing on ZFP36L2 identifies an important post-transcriptional regulator of steroid hormone metabolism. These results have the potential to impact adrenal diseases, specifically form of primary hyperaldosteronism. Finally, we expect to submit two manuscripts as a result of this work before the end of the calendar year.
Target groups:
Our research affects numerous target groups. Inappropriate production of hormone produced in the adrenal cortex causes major physiological disorders given the potent nature of these hormones. This includes diseases based on the overproduction of adrenal cortisol such as Cushing’s syndrome. Another major category is Conn’s syndrome and other forms of primary aldosteronism, which may impact up to 20% of hypertensive population. Due the gender differences in the presentation of primary aldosteronism, this work has the potential to more greatly benefit female health. This is important due to reported gender imbalance in animal studies and human clinical trials resulting in less female subjects.
