PROJECTS

Human Steroidogenesis

The human adrenal cortex synthesizes steroid hormones including glucocorticoids, mineralocorticoids and androgens. Defects resulting in either under- or over-production of these steroid hormones are pathological. For example, over production of aldosterone is responsible for ~%10 of hypertensive individuals (1⁄3 of all individuals). Our goal is to achieve a quantitative understanding of the dynamic molecular mechanisms regulating steroidogenesis. Ultimately, we hope to precisely modulate hormone production through potentiating or inhibiting RNA regulatory interactions. These approaches could have important implications for patients with adrenal disease.

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RBPs?

lncRNAs?

Ongoing projects in the lab focus on:
  1. The RNA regulatory dynamics underlying steroid production.
  2. The molecular mechanism of trans-acting regulatory factors controlling steroidogenesis, specifically:
    • RNA-binding proteins (RBPs)
    • Long non-coding RNAs (lncRNAs)
RNA regulatory dynamics

We performed an RNA-seq time course in responst to stimualtion with forskolin. We identified 4 distinct temporal responses to steroidogenesis. We want to understand what are the RNA-regulatory elements and factors that control the dynamics of the gene expression responses. Indeed, the genes within these clusters have different RNA stabilities (t1/2) and enrichment of regulatory elements within their 3’ UTRs.

RNA regulatory factors

RNA-binding proteins

Through siRNA screening we have identified numerous RBP that control hormone production. Our goal is to map the interaction sites for these RBPs and reveal the mechanisms by which regulate target RNAs.

Long non-coding RNA

We are focusing on two lncRNAs and the mechanisms by which they regulate steroidogenesis. We will employ novel approaches to map interactions between lncRNAs and proteins, as well as, DNA and RNA. 

RNA sequencing

Our goal is to improve multi-plexed RNA sequencing methods by streamlining the protocol, optimizing the efficiency of ligation reactions to lower RNA input requirements. Additionally, our cloning strategy would be broadly applicable to many assays in to investigate RNA regulation even for precious samples, including ribosome profiling, small RNA-seq, PAR-CLIP, 4sU-seq.

 

Multi-plexed/purpose Strategy

Cartoon schematic of library preparation:

  1. Three samples of fragmented, 5’-phosphorylated total RNA

  2. Simultaneous ligation of 3’-tag (red, orange, or yellow) and 5’ adapter (dark gray) to RNA samples by T4 RNA Ligase 2

  3. Pooled tagged-RNA samples and targeted depletion of rRNA (not shown)

  4. cDNA synthesis

  5. Amplification with P5 and P7 Illumina-barcoded primers

  6. Final library.