ORCID Profile
0000-0002-2441-8095
Current Organisation
University of Oxford
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Publisher: Cold Spring Harbor Laboratory
Date: 15-12-2017
DOI: 10.1101/234930
Abstract: Loss-of-function (LOF) methods, such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing, provide unparalleled power for studying the biological function of genes of interest. When coupled with transcriptomic analyses, LOF methods allow researchers to dissect networks of transcriptional regulation. However, a major concern is nonspecific targeting, which involves depletion of transcripts other than those intended. The off-target effects of each of these common LOF methods have yet to be compared at the whole-transcriptome level. Here, we systematically and experimentally compared non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible offtarget effects in gene expression, with CRISPRi exhibiting clonal variation in the transcriptional profile. As an illustrative ex le, we evaluated the performance of each method for deciphering the role of a long noncoding RNA (lncRNA) with unknown function. Although all LOF methods reduced expression of the candidate lncRNA, each method yielded different sets of differentially expressed genes upon knockdown as well as a different cellular phenotype. Therefore, to definitively confirm the functional role of a transcriptional regulator, we recommend the simultaneous use of at least two different LOF methods and the inclusion of multiple, specifically designed negative controls.
Publisher: Oxford University Press (OUP)
Date: 06-2018
DOI: 10.1093/NAR/GKY437
Publisher: Cold Spring Harbor Laboratory
Date: 19-07-2019
DOI: 10.1101/709030
Abstract: Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. The role of long noncoding RNAs (lncRNAs) in controlling these processes however remains largely unexplored. To identify lncRNAs with mitotic functions, we performed a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs. By investigating major hallmarks of cell ision such as chromosome segregation, mitotic duration and cytokinesis, we discovered numerous lncRNAs with functions in each of these processes. The chromatin-associated lncRNA, linc00899, was selected for in-depth studies due to the robust mitotic delay observed upon its depletion. Transcriptome analysis of linc00899 -depleted cells together with gain-of-function and rescue experiments across multiple cell types identified the neuronal microtubule-binding protein, TPPP 25, as a target of linc00899 . Linc00899 binds the genomic locus of TPPP 25 and suppresses its transcription through a cis -acting mechanism. In cells depleted of linc00899, the consequent upregulation of TPPP 25 alters microtubule dynamics and is necessary and sufficient to delay mitosis. Overall, our comprehensive screen identified several lncRNAs with roles in genome stability and revealed a new lncRNA that controls microtubule behaviour with functional implications beyond cell ision.
Publisher: Springer Science and Business Media LLC
Date: 15-04-2020
DOI: 10.1038/S41467-020-14978-7
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 ision 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 25 , as a target of linc00899 . We further show that linc00899 binds TPPP 25 and suppresses its transcription. In cells depleted of linc00899 , upregulation of TPPP 25 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 ision.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Fanni Gergely.