Polysome-profiling in small tissue samples

Author(s):Show less -

Liang, Shuo ^{[1, 2]} ; Bellato, Hermano Martins ; Lorent, Julie ^[2] ; Lupinacci, Fernanda C. S. ^[1] ; Oertlin, Christian ^[2] ; van Hoef, Vincent ^[2] ; Andrade, Victor P. ^[3] ; Roffe, Martin ^[1] ; Masvidal, Laia ^[2] ; Hajj, Glaucia N. M. ^[1] ; Larsson, Ola ^[2]

Affiliation

^[1] AC Camargo Canc Ctr, Int Res Ctr, Sao Paulo - Brazil

^[2] Karolinska Inst, Sci Life Lab, Dept Oncol Pathol, Stockholm - Sweden

^[3] AC Camargo Canc Ctr, Dept Pathol, Sao Paulo - Brazil

• Document type: Journal article
• Source: Nucleic Acids Research; v. 46, n. 1 JAN 9 2018.
• Web of Science Citations: 5

Abstract

Polysome-profiling is commonly used to study translatomes and applies laborious extraction of efficiently translated mRNA (associated with > 3 ribosomes) from a large volume across many fractions. This property makes polysome-profiling inconvenient for larger experimental designs or samples with low RNA amounts. To address this, we optimized a non-linear sucrose gradient which reproducibly enriches for efficiently translated mRNA in only one or two fractions, thereby reducing sample handling 5-10-fold. The technique generates polysomeassociated RNA with a quality reflecting the starting material and, when coupled with smart-seq2 singlecell RNA sequencing, translatomes in small tissues from biobanks can be obtained. Translatomes acquired using optimized non-linear gradients resemble those obtained with the standard approach employing linear gradients. Polysome-profiling using optimized non-linear gradients in serum starved HCT-116 cells with or without p53 showed that p53 status associates with changes in mRNA abundance and translational efficiency leading to changes in protein levels. Moreover, p53 status also induced translational buffering whereby changes in mRNA levels are buffered at the level of mRNA translation. Thus, here we present a polysome-profiling technique applicable to large study designs, primary cells and frozen tissue samples such as those collected in biobanks. (AU)

• FAPESP's process: 15/15451-3 - Study on the Regulation and Function of the RSK Family in Glioblastomas
• Grantee: Martín Roffé
• Support type: Regular Research Grants
• FAPESP's process: 13/03315-2 - Translatomic applied to the discovery of molecular alterations in gliomas
• Grantee: Fernanda Cristina Sulla Lupinacci
• Support type: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 14/04513-5 - Identification of molecular alterations in breast tumors: a translational approach
• Grantee: Hermano Martins Bellato
• Support type: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 14/15550-9 - Translational control in cancer
• Grantee: Glaucia Noeli Maroso Hajj
• Support type: Regular Research Grants