A single-copy knockin translating ribosome immunoprecipitation toolkit for tissue-specific profiling of actively translated mRNAs in C. elegans.

ABSTRACT: Here, we introduce a single-copy knockin translating ribosome immunoprecipitation (SKI TRIP) toolkit, a collection of Caenorhabditis elegans strains engineered by CRISPR in which tissue-specific expression of FLAG-tagged ribosomal subunit protein RPL-22 is driven by cassettes present in single copy from defined sites in the genome. Through in-depth characterization of the effects of the FLAG tag in animals in which endogenous RPL-22 has been tagged, we show that it incorporates into actively translating ribosomes and efficiently and cleanly pulls down cell-type-specific transcripts. Importantly, the presence of the tag does not impact overall mRNA translation, create bias in transcript use, or cause changes to fitness of the animal. We propose SKI TRIP use for the study of tissue-specific differences in translation and for investigating processes that are acutely sensitive to changes in translation like development or aging.

SUBMITTER: Wester LE 

PROVIDER: S-EPMC10088236 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature