Nutrient stress diverts RRN3 from rRNA transcription to alternative polyadenylation of autophagy mRNAs in ovarian cancer [CRISPR Screen]
Stress-induced alternative processing of mRNA is emerging as an essential mechanism to drive almost every hallmark of cancer. Through a genome-wide screening based on an abnormal transcriptional readthrough event favoring the malignant progression of ovarian carcinoma (OC), we identified novel mRNA processing regulators including RRN3, an essential factor for the transcriptional initiation of rRNA. The long-read RNA sequencing and PAR-CLIP analyses revealed that RRN3 was involved in the usage of alternative polyadenylation (APA) sites, resulting in the altered stability of autophagy-related mRNAs. More interestingly, we discovered that nutrient-deprivation-induced phosphorylation of RRN3 at serine 199 was sufficient to divert RRN3 out of the nucleolus to the nuclear plasma, where RRN3 regulated the APA of autophagy mRNAs, such as OPTN, to enhance their stability and eventually promoted autophagy. Further in vivo experiments showed that nutrient-stress-triggered switch of RRN3 from rRNA transcription to APA regulation was essential for the growth and dissemination of OC in mice. Overall design: The GeCKO (v2.0) CRISPR library made by Zhangfeng's lab was purchased from Addgene and amplified using the recommended protocol. The library contains 122,411 sgRNAs targeting 19,050 protein-encoding genes, 1,864 miRNA and 1,000 non-targeting control sgRNAs. A dual-fluorescence transcriptional readthrough (TRT) reporter system was established using pLV-EF1a-MCS-IRES-Bsd vector for the establishment of stable SK-OV-3 cells harboring the TRT reporter sequence (SK-OV-3TRT), in which the majority encoding sequences of COMMD3 and BMI1 were replaced by the mCherry- and EGFP-encoding sequences, respectively, and the sequence in between (including the terminal part of exon 7, intron 7, exon 8 of COMMD3, and the exon 1, intron 1, initial part of exon 2 of BMI1) was kept in frame. Lentivirus carrying the library were prepared according to the manufacturer's protocol and infected the SK-OV-3TRT cells. The viral titer was estimated and the transduction was performed at a low multiplicity of infection (MOI) around 0.3. After puromycin (2 µg/mL) selection, mCherry+ EGFP- cells were sorted by flow cytometry. The sorted cells (mCherry+ EGFP-) and the parental cells (unsorted cells) were subjected to genomic DNA extraction, PCR-based amplification of sgRNA-encoding sequences and the high-throughput DNA sequencing