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Identification of VIPP1-interacting proteins in Arabidopsis.

ABSTRACT: Results from co-immunoprecipitation (co-IP)–MS analysis of VIPP-YFP bait, expressed in wild-type Arabidopsis thaliana. GFP-Trap magnetic agarose beads were used for co-IP. Co-precipitated peptides were digested on the beads and analyzed by liquid chromatography–mass spectrometry (LC-MS). The MS data were processed using the Philosopher toolkit, with MSFragger as the search engine, followed by IonQuant. SAINTexpress software was used to score potential interactions between the observed proteins (potential prey) and the bait protein. It is recommended to use .raw files for reanalysis.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Arabidopsis Thaliana (ncbitaxon:3702)

SUBMITTER: Mayank Sharma

PROVIDER: MSV000099084 | MassIVE |

SECONDARY ACCESSION(S): PXD068188

REPOSITORIES: MassIVE

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Project description:Potential miR-202 targets were identified using a targetome-wide RIP-based microarray. HeLa cells were transfected with either a miR-202 mimic or a scrambled single-stranded RNA negative control. RNA was isolated from total cell lysate prior to IP and from antibody-immobilized Protein G agarose beads-RNP complexes (post-IP). Ribonucleoprotein IP was performed using the RIP-Assay kit for microRNA (MBL) according to the protocol described by the manufacturer. Briefly, anti-EIF2C2/Ago2 monoclonal antibody (Novus Biologicals, LLC) was incubated with Protein G plus agarose beads (Pierce) at 4M-BM-0C overnight to prepare antibody-immobilized beads. 20 million cells were harvested and washed four times with ice-cold DEPC-treated PBS. The cell pellet was lysed with 500 M-NM-<l of lysis buffer and the supernatant was incubated with Protein G agarose beads without antibody to reduce nonspecific adsorption. The cell lysate was then transferred into a tube containing antibody-immobilized Protein G agarose beads and incubated for 3 hours at 4M-BM-0C. Genome-wide expression levels were analyzed in total RNA samples from total cell lysate and antibody-immobilized Protein G agarose beads-RNP complexes from cells transfected with miR-202 mimic or negative control using the Agilent 44K 60-mer human whole-genome microarray. Signal hybridization and scans were performed by MOGene, LC (St Louis, MO) (run in biological duplicate). The normalized signal intensities of probes from RNP-bead complexes (post-IP fraction) were divided by the signal intensities from total cell lysate (pre-IP fraction) in miR-202 mimic-transfected cells. Transcripts were identified as members of the global miRNA targetome if they exhibited an enrichment fold change > 2.0. From this list, post-IP to pre-IP signal intensity ratios in miR-202 mimic-transfected cells were divided by post-IP to pre-IP signal intensity ratios in NC cells. Transcripts exhibiting normalized signal ratios of > 1.5 were considered to be bound with miR-202 in the RNA-induced silencing complex (RISC), and therefore potential direct miR-202 targets.

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Identification of VIPP1-interacting proteins in Arabidopsis.

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