Details

Autor(en) / Beteiligte

• Dinkeloo, Kasia
• Pelly, Zoe
• McDowell, John M.
• Pilot, Guillaume

Titel

A split green fluorescent protein system to enhance spatial and temporal sensitivity of translating ribosome affinity purification

Ist Teil von

• The Plant journal : for cell and molecular biology, 2022-07, Vol.111 (1), p.304-315

Ort / Verlag

England: Blackwell Publishing Ltd

Erscheinungsjahr

2022

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• SUMMARY Translating ribosome affinity purification (TRAP) utilizes transgenic plants expressing a ribosomal protein fused to a tag for affinity co‐purification of ribosomes and the mRNAs that they are translating. This population of actively translated mRNAs (translatome) can be interrogated by quantitative PCR or RNA sequencing. Condition‐ or cell‐specific promoters can be utilized to isolate the translatome of specific cell types, at different growth stages and/or in response to environmental variables. While advantageous for revealing differential expression, this approach may not provide sufficient sensitivity when activity of the condition/cell‐specific promoter is weak, when ribosome turnover is low in the cells of interest, or when the targeted cells are ephemeral. In these situations, expressing tagged ribosomes under the control of these specific promoters may not yield sufficient polysomes for downstream analysis. Here, we describe a new TRAP system that employs two transgenes: One is constitutively expressed and encodes a ribosomal protein fused to one fragment of a split green fluorescent protein (GFP); the second is controlled by a stimulus‐specific promoter and encodes the second GFP fragment fused to an affinity purification tag. In cells where both transgenes are active, the purification tag is attached to ribosomes by bi‐molecular folding and assembly of the split GFP fragments. This approach provides increased sensitivity and better temporal resolution because it labels pre‐existing ribosomes and does not depend on rapid ribosome turnover. We describe the optimization and key parameters of this system, and then apply it to a plant–pathogen interaction in which spatial and temporal resolution are difficult to achieve with current technologies. Significance Statement Translating ribosome affinity purification (TRAP) has been modified to allow with increased sensitivity the isolation of RNA from sets of cells in which the activity of condition/cellspecific promoters is weak, ribosome turnover is low, or cells whose nature is ephemeral. Based on the use of a split linker constituted of the GFP driven by a pathogen‐inducible promoter, this new TRAP system enabled efficient isolation of translated RNA from pathogen‐infected leaf cells.