Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription

Abstract

The steroidogenic acute regulatory protein (StAR) has been proposed to serve as the switch that can turn on/off steroidogenesis. We investigated the events that facilitate dynamic StAR transcription in response to cAMP stimulation in MA-10 Leydig cells, focusing on splicing anomalies at StAR gene loci. We used 3’ reverse primers in a single reaction to respectively quantify StAR primary (p-RNA), spliced (sp-RNA/mRNA), and extended 3’ untranslated region (UTR) transcripts, which were quantitatively imaged by high-resolution fluorescence in situ hybridization (FISH). This approach delivers spatio-temporal resolution of initiation and splicing at single StAR loci, and transfers individual mRNA molecules to cytoplasmic sites. Gene expression was biphasic, initially showing slow splicing, transitioning to concerted splicing. The alternative 3.5-kb mRNAs were distinguished through the use of extended 3’ UTR probes, which exhibited distinctive mitochondrial distribution. Combining quantitative PCR and FISH enables imaging of localization of RNA expression and analysis of RNA processing rates.

A single cell level measurement of StAR expression and activity in adrenal cells

“…We suggest that each cell is at a different stage of endogenous stimulation as marked by this ratio. These cell differences are consistent with the asymmetric activation of StAR loci and asynchronous responses of individual cells previously reported for MA-10 cells (Lee et al, 2016a; Lee et al, 2015). …”

“…However, the absence of increases in either exon 7 or spliced transcripts for 15 minutes indicates a pause in pol2 elongation at this point, which is coupled to splicing. Very similar delays are apparent in both Y-1 and MA-10 cells (Lee et al, 2016a; Lee et al, 2015). This delay in elongation delivers spatially resolved clusters of unspliced and spliced transcripts that can be visualized at the loci with the Nikon’s structured illumination microscope (N-SIM) (Figure 2A).…”

“…This basal expression has been addressed by a combination of q-PCR and high-resolution fluorescence in situ hybridization (HR-FISH) microscopy described in detail elsewhere (Lee et al, 2016a). …”

“…Thus, individual loci are activated on different time courses while single cells become engaged separately over the course of about 60 minutes(Lee et al, 2016a). Early and late onset processes have been distinguished in MA-10 cells.…”

ACTH Action on StAR Biology

“…The model that AKAP1 localizes PKAR2 at the OMM provides a platform for localized phosphorylation of StAR (Grozdanov and Stocco, 2012; Merrill and Strack, 2014, Figure 1). Recently StAR mRNA association with mitochondria in MA-10 cells was demonstrated using high resolution fluorescence in situ hybridization (Lee et al, 2016a). Key findings from this work indicate that StAR mRNA-mitochondrial interactions are dynamic, and suggest that when StAR transcription is maximally activated, newly transcribed and processed StAR mRNA is delivered to the cytoplasm and mitochondria for efficient translation.…”