Dataset Information

TurboID-mediated proximal labeling of Drosophila caspase Drice in adult brains

ABSTRACT: V5-TurboID was knock-in tagged in Drosophila caspase Drice which is expressed in adult brains. Flies were fed with 100 µM biotin for protein biotinylation in two biological replicates. Biotinylated protein-containing lysate extracted from adult brains was subjected to FG-NeutrAvidin beads purification followed by on-beads trypsin digestion. Digested peptides were desalinated and purified using GL-tip SDB. Purified peptides were subjected to LC-MS/MS analysis.

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

SUBMITTER: Masayuki Miura

PROVIDER: PXD042922 | JPOST Repository | Tue Feb 06 00:00:00 GMT 2024

REPOSITORIES: jPOST

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			Action	DRS
	20210215Hanawa03.raw	Raw
	20210215Hanawa05.raw	Raw
	20210215Hanawa07.raw	Raw
	20210215Hanawa09.raw	Raw
	20210215_DriceV5TurboID_normalized.pdResult	Other

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Project description:Circadian behaviors are regulated by intrinsic biological clocks consisting of central molecular oscillators and output pathways. Despite significant progress in elucidating the central timekeeping mechanisms, the molecular pathways coupling the circadian pacemaker to overt rhythmic behavior and physiology remain elusive. The Drosophila LARK RNA-binding protein is a candidate for such a coupling factor. Previous research indicates that LARK functions downstream of the clock to mediate behavioral outputs. To better understand the roles of LARK in the Drosophila circadian system, we sought to identify RNA molecules associated with LARK in vivo, using a novel strategy that involves capturing the RNA ligands by immunoprecipitation, visualizing the captured RNAs using whole gene microarrays, and identifying functionally relevant targets through genetic screens. Experiment Overall Design: LARK-containing ribonucleoprotein complexes (LARK-RNPs) were precipitated from lysates of hand-dissected pharate adult brains using an affinity-purified anti-LARK antibody (around 1000 brains were used per immunoprecipitation experiment). A portion of each lysate was saved prior to immunoprecipitations (IPs) in order to measure the relative abundance of transcripts in a total RNA sample. RNAs extracted from the LARK-RNP and total RNA samples were labeled and hybridized to Drosophila whole-genome gene microarrays; signal intensities for individual genes were compared between samples to identify those RNAs that were enriched by immunoprecipitation (relative to their abundances in total RNA). RNAs that were selectively enriched in the LARK-RNP samples were considered to be potential targets of the RNA-binding protein. Experiment Overall Design: Due to the difficulty to dissect large amount of fly brains, only two such immunoprecipitation experiments were performed, each generating an IP RNA sample and a total RNA (control) sample. The amount of RNAs obtained from IP is very small thus only one array is used for each sample - i.e. there are only biological replicates and no technical replicate.

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TurboID-mediated proximal labeling of Drosophila caspase Drice in adult brains

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