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Effect of PCIF1 knockdown on gene exppression in HeLa S3 cells

ABSTRACT: We previously identified PCIF1 (Phosphorylated CTD Interacting Factor 1) as a novel phosphorylated C-terminal domain (CTD) of RNA polymerase II. We also recently identified PCIF1 as a new cap-specific adenine N6 methyltransferase (CAPAM) responsible for creating N6, 2’-O-dimethyladenosine (m6Am) at the 5’-end of mRNAs. However, it remains unclear how PCIF1 regulates gene expression. To identify genes whose expression levels are affected by PCIF1 knockdown in human cultured cells, we performed gene expression profiling by microarray analysis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE156768 | GEO | 2023/09/30

REPOSITORIES: GEO

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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.