Polysome Fractionation to Analyze mRNA Distribution Profiles

• Published in: Bio-protocol Vol. 7; no. 3
• Main Authors: Panda, Amaresh C, Martindale, Jennifer L, Gorospe, Myriam
• Format: Journal Article
• Language: English
• Published: United States Bio-Protocol 05.02.2017; Bio-protocol LLC
• Subjects: Biology; Methods; Fractionation; Sucrose gradient; Ribosome; mRNA translation; Protein synthesis; Polysomes; RT-qPCR
• ISSN: 2331-8325; 2331-8325
• DOI: 10.21769/BioProtoc.2126

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 , 2014a and 2014b; Abdelmohsen , 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 , 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.