A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division

Abstract

Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. With a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs, we identify numerous lncRNAs involved in key steps of cell division such as chromosome segregation, mitotic duration and cytokinesis. Here, we provide evidence that the chromatin-associated lncRNA, linc00899, leads to robust mitotic delay upon its depletion in multiple cell types. We perform transcriptome analysis of linc00899-depleted cells and identify the neuronal microtubule-binding protein, TPPP/p25, as a target of linc00899. We further show that linc00899 binds TPPP/p25 and suppresses its transcription. In cells depleted of linc00899, upregulation of TPPP/p25 alters microtubule dynamics and delays mitosis. Overall, our comprehensive screen uncovers several lncRNAs involved in genome stability and reveals a lncRNA that controls microtubule behaviour with functional implications beyond cell division.

Functional Screening Techniques to Identify Long Non-Coding RNAs as Therapeutic Targets in Cancer

“…Unlike siRNAs, shRNAs can integrate into genomic DNA, allowing for longer-term repression of target genes [ 66 , 67 ]. A number of high-throughput RNAi screens targeting lncRNAs for functional analysis in cancer have been performed over the past decade [ 68 , 69 , 70 , 71 , 72 , 73 , 74 ]. We will discuss the three most recent ones below.…”

“…Negative control siRNAs, siRNAs targeting the protein-coding gene Ch-TOG/CKAP5 , and cells without any siRNA treatment were used as controls [ 69 ]. Two RNAi screens were performed in parallel (Screens A and B) using antibodies to label microtubule cytoskeleton, centrosomes and nuclei [ 69 ]. Screen A additionally labelled the actin cytoskeleton and Screen B labelled mitotic cells [ 69 ].…”

“…Two RNAi screens were performed in parallel (Screens A and B) using antibodies to label microtubule cytoskeleton, centrosomes and nuclei [ 69 ]. Screen A additionally labelled the actin cytoskeleton and Screen B labelled mitotic cells [ 69 ]. The cells were fixed after 48 h and processed for immunostaining and image acquisition [ 69 ].…”

“…Screen A additionally labelled the actin cytoskeleton and Screen B labelled mitotic cells [ 69 ]. The cells were fixed after 48 h and processed for immunostaining and image acquisition [ 69 ]. Automated image analysis was used to segment the cells followed by quantification of mitotic progression, chromosome segregation and cytokinesis using an in-house developed pipeline [ 69 ].…”

“…The cells were fixed after 48 h and processed for immunostaining and image acquisition [ 69 ]. Automated image analysis was used to segment the cells followed by quantification of mitotic progression, chromosome segregation and cytokinesis using an in-house developed pipeline [ 69 ]. Subsequent validation targeting their top 25 lncRNA candidates confirmed their findings [ 69 ].…”