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Identification of DYRK3 interaction partners by immunoprecipitation-MS and TurboID

ABSTRACT: Proteins that interact with overexpressed GFP-DYRK3 were pulled down for identification by LC-MS/MS. Interaction strength and likelihood were calculated by comparison to pulldown of GFP alone. Data were compared to Turbo-ID proximity labelling data for DYRK3 in HeLa cells to identify consistently associated proteins. These included many secretory proteins. Further experiments established that DYRK3 phosphorylates SEC16A to regulate the liquid-liquid phase separation of ER exit sites.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293t Cell, Hela Cell

SUBMITTER: Katrina Meyer

LAB HEAD: Lucas Pelkmans

PROVIDER: PXD041550 | Pride | 2023-06-23

REPOSITORIES: Pride

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Project description:Our computational analyses of sequencing depth and the discovery rates of sequence elements in the 3M-bM-^@M-^YUTR strongly demonstrate that interrogation of the 3M-bM-^@M-^YUTRome in specific tissues and cell types across development will greatly expand the identification of new 3M-bM-^@M-^YUTR isoforms and the sequence elements therein. Therefore, we will generate and sequence the 3M-bM-^@M-^YUTRs from the cell types isolated from the developing germline, mature germ cells, and early embryogenesis. In addition, we will identify the 3M-bM-^@M-^YUTRs for the transcripts isolated from the major somatic tissues during late- and post-embryonic development. Based on our sequencing estimates, we anticipate that the tissue-specific interrogation of the 3M-bM-^@M-^YUTRome will reveal a far greater diversity of novel 3M-bM-^@M-^YUTR isoform expression that is masked in the whole-worm 3M-bM-^@M-^YUTRome sequence data. Using the transgenic myo-3::PABP strain, we have generated polyA-captured libraries for late embryos and across the major stages of post-embryonic development for the muscle transcriptome. We propose to generate these polyA-captured libraries for transcripts expressed in the other major tissues across development. PolyA-captured libraries will be generated for deep sequencing following the tissue-specific isolation of mRNAs by PABP pulldowns (the PABP immunoprecipitations will take advantage of a FLAG epitope which is fused to PABP in all of the transgenic strains). We propose to validate the expression of tissue- specific transcripts by qPCR for select transcripts known to be expressed in those particular tissues. In addition, following the strategy we have employed for the large-scale polyA-captured libraries from muscle, we will perform quality checks for 3M-JM-< end capture by manually sequencing ~60 clones isolated from each library. Once we have obtained the deep sequencing data, we will analyze all of the sequence reads using the bioinformatics pipeline that we established for the whole-worm polyA- captured sequences (Mangone et al., 2010). Four samples representing gravid, L2, L3, and L4 developmental stages were analyzed.

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Identification of DYRK3 interaction partners by immunoprecipitation-MS and TurboID

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