Analysis of translation using polysome profiling.
During the past decade, there has been growing interest in the role of translational regulation of gene expression in many organisms. Polysome profiling has been developed to infer the translational status of a specific mRNA species or to analyze the translatome, i.e. the subset of mRNAs actively translated in a cell. Polysome profiling is especially suitable for emergent model organisms for which genomic data are limited. In this paper, we describe an optimized protocol for the purification of sea urchin polysomes and highlight the critical steps involved in polysome purification. We applied this protocol to obtain experimental results on translational regulation of mRNAs following fertilization. Our protocol should prove useful for integrating the study of the role of translational regulation in gene regulatory networks in any biological model. In addition, we demonstrate how to carry out high-throughput processing of polysome gradient fractions, for the simultaneous screening of multiple biological conditions and large-scale preparation of samples for next-generation sequencing.
CNRS, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, Roscoff cedex, France.|Sorbonne Universités, UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, Roscoff cedex, France.
Héloïse
H
Chassé
CNRS, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, Roscoff cedex, France.|Sorbonne Universités, UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, Roscoff cedex, France.
Sandrine
S
Boulben
CNRS, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, Roscoff cedex, France.|Sorbonne Universités, UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, Roscoff cedex, France.
Vlad
V
Costache
CNRS, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, Roscoff cedex, France.|Sorbonne Universités, UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, Roscoff cedex, France.
Patrick
P
Cormier
CNRS, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, Roscoff cedex, France.|Sorbonne Universités, UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, Roscoff cedex, France.
Julia
J
Morales
D000818:Animals; D005260:Female; D005306:Fertilization / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D008297:Male; D047328:Paracentrotus / Q000196:embryology / Q000235:genetics / Q000502:physiology; D011132:Polyribosomes / Q000235:genetics* / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA
D012333:RNA, Messenger