Polysome Profiling
Polysome-seq involves the separation and collection of transcripts into highly and lowly translated fractions. Next-generation sequencing of these transcripts reveals the full-length information of translating mRNAs which can be used to investigate post-transcriptional control of gene expression (1).
Unlike RIBO-seq, where the short read lengths of ribosome protected fragments prevent accurate isoform delineation, polysome-seq allows for the sequencing of long read lengths which enables precise determination of transcript isoforms, splice variants and fusion genes that are actively undergoing translation (2).
Polysome-seq also allows for the targeted selection and sequencing of specific transcripts of interest, providing exquisite sensitivity for transcript detection.
Overview: Polysomes are separated on a sucrose gradient based on the number of ribosomes on transcripts (A). A light fraction (monosomes) and a heavy fraction (Polysomes with 3 or more ribosomes per transcript) are collected (B). The RNA from each fraction is purified, poly-A selected and fragmented. mRNA Fragments are converted into a cDNA library which undergoes deep sequencing.
Applications
- Determine the degree to which alternative splice isoforms are translated.
- Evaluate the translation of oncogenic fusion genes as viable targets for cancer diagnosis and treatment.
- Measure the translational activity of targeted transcripts, from traditional drug targets to mRNA therapeutics.
- Identify novel regulatory elements such as cis- or trans-acting elements that regulate mRNA translation.
- Identify shifts in ribosome density between conditions at the transcript level.
Polysome profiling vs Ribosome profiling
- Both polysome profiling and ribosome profiling can be used to study global translational activity, but each has distinct strengths.
- Polysome profiling measures ribosome density of each mRNA, providing a direct measurement of translational activity.
- Ribosome profiling captures positional information of ribosome footprints at the subcodon level and is more suitable for investigating alternative start codons or open reading frames.
- Ribosome profiling can reveal codon-specific regulation of gene expression during the translational elongation stage.
- The size of ribosome footprint, which is unique to ribosome profiling, may indicate differential conformation of ribosomes or the presence of disomes.
References
1. Pereira, I.T., Spangenberg, L., Robert, A.W., Amorin, R., Stimamiglio, M.A., Naya, H. and Dallagiovanna, B. (2018). Polysome profiling followed by RNA-seq of cardiac differentiation stages in hESCs. Sci Data. 4(5):180287.
2. Floor, S.N. and Doudna, J.A. (2016). Tunable protein synthesis by transcript isoforms in human cells. Elife. 6(5):e10921.
3. Jin HY, Xiao C. An Integrated Polysome Profiling and Ribosome Profiling Method to Investigate In Vivo Translatome. Methods Mol Biol. 2018;1712:1-18.