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

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dc.contributor.authorDinkeloo, Kasiaen
dc.contributor.authorPelly, Zoeen
dc.contributor.authorMcDowell, John M.en
dc.contributor.authorPilot, Guillaumeen
dc.date.accessioned2022-06-14T12:49:47Zen
dc.date.available2022-06-14T12:49:47Zen
dc.date.issued2022-04-18en
dc.description.abstractTranslating 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.en
dc.description.versionPublished versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1111/tpj.15779en
dc.identifier.eissn1365-313Xen
dc.identifier.issn0960-7412en
dc.identifier.pmid35436375en
dc.identifier.urihttp://hdl.handle.net/10919/110772en
dc.language.isoenen
dc.publisherWileyen
dc.rightsCreative Commons Attribution-NonCommercial-NoDerivatives 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/en
dc.subjecttranslationen
dc.subjectribosomeen
dc.subjectArabidopsisen
dc.subjectHyaloperonospora arabidopsidisen
dc.subjecttechnical advanceen
dc.titleA split green fluorescent protein system to enhance spatial and temporal sensitivity of translating ribosome affinity purificationen
dc.title.serialPlant Journalen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

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