Abstract
Localization of RNAs to various subcellular destinations has emerged as a widely used mechanism that regulates a large proportion of transcripts in polarized cells. A number of methodologies have been developed that allow detection and imaging of RNAs at single-molecule resolution. However, methodologies to quantitatively describe RNA distributions are limited. Such approaches usually rely on the identification of cytoplasmic and nuclear boundaries which are used as reference points. Here, we describe an automated, interactive image analysis program that facilitates the accurate generation of cellular outlines from single cells and the subsequent calculation of metrics that quantify how a population of RNA molecules is distributed in the cell cytoplasm. We apply this analysis to mRNAs in mouse and human cells to demonstrate how these metrics can highlight differences in the distribution patterns of distinct RNA species. We further discuss considerations for the practical use of this tool. This program provides a way to facilitate and expedite the analysis of subcellular RNA localization for mechanistic and functional studies.
Highlights
RNA molecules are transcribed in the nucleus and exported to the cytoplasm where they usually serve as messengers for decoding the genetic information into protein products
RDI analysis can quantitatively differentiate distinct RNA distributions in cell populations. To illustrate how this analysis can be used to describe the distribution of distinct RNA species, we used single molecule in situ hybridization to detect mRNAs in mouse fibroblast cells
We visualized RNAs that exhibit distinct distribution patterns (Fig. 3A,B). These include: (1) the P4hb mRNA, encoding the beta subunit of prolyl-4-hydroxylase, which functions as an ER chaperone[27], (2) the Arpc[3] mRNA, encoding subunit 3 of the actin-nucleating Arp2/3 complex[28], (3) the RhoA mRNA, encoding the RhoA GTPase involved in organization of the actin cytoskeleton[29], and (4) the Cyb5r3 mRNA, encoding cytochrome b5 reductase 3 involved in fatty acid and cholesterol metabolism[30]
Summary
RNA molecules are transcribed in the nucleus and exported to the cytoplasm where they usually serve as messengers for decoding the genetic information into protein products. In highly polarized cells, such as neurons, quantitative assessment of RNA localization from imaging data can be done by assigning RNAs into distinguishable compartments such as dendrites, axonal shafts or growth cones[8,22] Such distinctions become much harder and inherently biased in smaller cell types which have either irregular and varied morphologies or lack clear boundaries of functional locations. There is no objective way of defining the exact spatial boundaries of the leading edge of a migrating cell or the apical and basal surfaces of an epithelial cell[11,12,16,23] Such demarcations apply a binary choice onto the description of RNA distributions, precluding the ability to detect and differentiate distributions which result from more gradual RNA concentration gradients. We further present practical considerations for the implementation of this tool
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