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. 2017 Feb 5;7(3):e2126.
doi: 10.21769/BioProtoc.2126.

Polysome Fractionation to Analyze mRNA Distribution Profiles

Amaresh C Panda  1 , Jennifer L Martindale  1 , Myriam Gorospe  1
Affiliations

Polysome Fractionation to Analyze mRNA Distribution Profiles

Amaresh C Panda et al. Bio Protoc. .

Erratum in

Abstract

Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al., 2014a and 2014b; Abdelmohsen et al., 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al., 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.

Keywords: Fractionation; Polysomes; Protein synthesis; RT-qPCR; Ribosome; Sucrose gradient; mRNA translation.

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Figures

Figure 1.
Figure 1.. Schematic of sucrose gradient preparation
Figure 2.
Figure 2.. Schematic of polysome fractionation
Figure 3.
Figure 3.. Effect of treatment on distribution of mRNA A across the sucrose gradient.
A. Cytoplasmic lysates from control and treated cells were fractionated through sucrose gradients. Global RNA polysome profiles generated by the density gradient fractionation system are shown. B. The relative distribution of the % mRNA A (left) and GAPDH mRNA (right), encoding a housekeeping protein, over the sucrose gradient was studied by RT-qPCR analysis of the RNA in each of the 12 gradient fractions.
See this image and copyright information in PMC

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