biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsVedran FrankeNextSeq 550RNA-seq of coding RNAHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14530Transcription and RNA processing are tightly coupled and precisely coordinated to ensure appropriate levels of mature transcripts. The C-terminal domain (CTD) of RNA polymerase II (Pol II) is phosphorylated differentially during the transcription cycle and serves as a landing pad for a variety of transcriptional regulators and RNA processing proteins. PHD finger protein 3 (PHF3) binds to the serine-2 phosphorylated Pol II CTD with its Spen Paralogue and Orthologue C-terminal (SPOC) domain and regulates transcription elongation and mRNA stability. Here we show that PHF3 binds target RNAs by recognizing a G-rich motif prone to form G-quadruplexes (G4s). Two PHF3 zinc finger domains, PHD (plant homeo domain) and TLD (TFIIS-like domain) act in concert to bind and destabilize target RNAs and their deletion in HEK293T cells causes massive deregulation of gene expression. Together these results establish PHF3 as a Pol II and an RNA-binding protein that coordinates transcription elongation with RNA decay to regulate neuronal gene expression.biostudies-arrayexpressSequencing - Sequencing was performed on an Illumina NextSeq 550 instrument in readmode SR75 by the Next Generation Sequencing facility at Vienna BioCenter Core Facilities (VBCF).Sample Collection - Cells from a 90% confluent 10 cm dish were harvested and counted, 8 million cells were mixed with 2 million Drosophila S2 cells as a spike-in control.Library Construction - rRNA was depleted using RiboCop rRNA depletion kit (Human/Mouse/Rat) (Lexogen) and libraries were prepared using Corall total RNA Seq kit (Lexogen) according to the manufacturer’s instructions, starting with 600 ng total RNA input.Nucleic Acid Extraction - Cell pellets from one well of a 6-well plate were resuspended in 1 mL TRI reagent (Sigma) and incubated for 5 min at room temperature. 200 µL chloroform (Applichem) was added and the lysate was vortexed and centrifuged at max. speed (21130 g) for 15 min at 4°C. The aqueous layer was transferred to a new tube and precipitated with 0.5 mL isopropanol. The RNA pellet was isolated by centrifugation for 30 min at 4°C, washed with 1 mL 75% ethanol, re-centrifuged for 10 min, dried and resuspended in 70 µL RNase free water. 20 µg RNA was treated with 40 U DNaseI (Roche) for 30 min at 37°C and subsequently purified by phenol-chloroform extraction and ethanol precipitation.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesDea SladeVedran FrankefalsePHF3 regulates RNA stability through PHD and TLD domains - RNAseq PHD - TLDTranscription and RNA processing are tightly coupled and precisely coordinated to ensure appropriate levels of mature transcripts. The C-terminal domain (CTD) of RNA polymerase II (Pol II) is phosphorylated differentially during the transcription cycle and serves as a landing pad for a variety of transcriptional regulators and RNA processing proteins. PHD finger protein 3 (PHF3) binds to the serine-2 phosphorylated Pol II CTD with its Spen Paralogue and Orthologue C-terminal (SPOC) domain and regulates transcription elongation and mRNA stability. Here we show that PHF3 binds target RNAs by recognizing a G-rich motif prone to form G-quadruplexes (G4s). Two PHF3 zinc finger domains, PHD (plant homeo domain) and TLD (TFIIS-like domain) act in concert to bind and destabilize target RNAs and their deletion in HEK293T cells causes massive deregulation of gene expression. Together these results establish PHF3 as a Pol II and an RNA-binding protein that coordinates transcription elongation with RNA decay to regulate neuronal gene expression.2025-12-04T00:00:00Z2025-12-04T02:01:49.489Z2024-10-10T14:07:39.775ZE-MTAB-14530ERP165009EFO_0002944EFO_0004170EFO_0005518EFO_0003738EFO_0004184