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Proteome data for Colorectal cancer cells transfected with different RNAs

ABSTRACT: Exon skipping (ES) events, known as the most prevalent form of alternative splicing, lead to the hallmark of tumorigenesis and considered to be therapeutic targets. To date, systematic functional interrogation of ES events in cancer are still largely unexplored. Here, we demonstrated the effectiveness of CRISPR-RfxCas13d in silencing transcripts derived from ES. Specifically, guide RNAs (gRNAs) targeting sequences spanning exon-skipping junction sites were designed and successfully employed without affecting their full-length counterparts. To comprehensively investigate ES events, we constructed a transcriptome-wide library of CRISPR-RfxCas13d gRNAs targeting 3,744 human ES events. We conducted loss-of-function screening both in vitro and in vivo using colorectal cancer (CRC) tumor cells. This approach led to the identification of several ES events crucial for cell growth. Notably, HMGN3 Δ6, a common oncogenic transcript derived from the ES of exon 6, exhibited a significant promotion of CRC proliferation. This work not only established CRISPR-RfxCas13d as a valuable tool for exploring and understanding ES events but also contributed to a comprehensive and large-scale characterization of the functional roles of ES events in CRC. The findings underscore the importance of ES events in CRC tumorigenesis, highlighting their potential as therapeutic targets.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Colon

DISEASE(S): Colon Cancer

SUBMITTER: Qiang Sun

LAB HEAD: Qiang Sun

PROVIDER: PXD062521 | Pride | 2025-06-12

REPOSITORIES: Pride

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Project description:N6,2'-O-dimethyladenosine (m6Am) is a critical and reversible RNA modification that plays a significant role in regulating RNA stability and gene expression. However, the lack of precise tools for manipulating m6Am modifications on specific transcripts has hindered the ability to elucidate the relationship between m6Am-modified transcripts and their phenotypic outcomes. Here, we present a CRISPR-Cas13d-based tool for the targeted installation of m6Am modifications on specific RNA transcripts. This tool is engineered by fusing a catalytically inactive variant of RfxCas13d (dCasRx) with the m6Am methyltransferase PCIF1. The dCasRx-PCIF1 fusion protein enables precise methylation of target m6Am-modified RNAs. We demonstrate that INSTALLER can effectively install m6Am modifications on GAPDH and ACTB RNAs, significantly enhancing their stability. MTD keywords Human, HEK293T cells MTD sample_processing_protocol Protein extraction and tryptic digestion. Cell lysates were lysed in lysis buffer (RPIA) supplemented with protease inhibitors and phosphatase inhibitors for 30 min. Then they were sonicated by grinding instrument at 4 ℃ and centrifuged at 12000 rpm for 15 min to remove the tissue debris. The protein in supernatants were collected and measured by using BCA protein assay. Next, 20 mM Tris (2-chloroethyl) phosphate (TCEP) was added to the protein solution to react at 60℃ for 1 h. After that, 200 mM Chloroacetamide (CAA) was added to the mixture and react at room temperature for 30 min in darkness. After the reaction, ice-cold acetone was used to precipitate the protein at -20℃ for 4 h. The pellet was air-dried and then resuspended in 150 μl 0.1M ABB. Protein samples underwent trypsin digestion (enzyme-to-substrate ratio of 1:25 at 37 ℃for 16 hours) followed by desalting through sola HRP cartridges and vacuum- dried by Speed Vac. Nano-LC-MS/MS analysis. Peptide samples were analyzed by an EASY-nLC 1200 LC system coupled with Orbitrap mass spectrometry (Thermo Fisher). The column was C18 nano-capillary analytical column (25 cm*75 mm, 1.9 μm). Peptides were re-dissolved in mobile phase A (Water/CAN/formic acid, 98/2/0.1, v/v). The gradient was 0-5 min, 5-10% B; 5-55 min, 10-28% B; 55-58 min, 28-40% B，58-63 min, 40-95% B; 63-75 min; 95% B with flow rate of 350 nL/min. Mass spectrometry was operated under a data independent acquisition mode (DIA). The m/z range was 350 to 1500 Da in MS1 with the resolution of 60,000. The automatic gain control (ACG) was set as 3e6. The maximal ion injection time was 50 ms. MS2 acquisition was higher energy collision dissociation (HCD) with the collision energy of 30 eV. The resolution was 30,000, with loop count set to: 25, MSX count set to: 1, isolation window set to: 8.3m/z, fixed maximum mass set to: 200m/z, respectively.

			Action	DRS
	DIANN_Expression_Res.tsv	Tabular
	HCT-116_HMGN3_FL1.mzML	Mzml
	HCT-116_HMGN3_FL2.mzML	Mzml
	HCT-116_HMGN3_FL3.mzML	Mzml
	HCT-116_HMGN3_S1.mzML	Mzml

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Proteome data for Colorectal cancer cells transfected with different RNAs

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