Dataset Information

Tumour mutations in long noncoding RNAs enhance cell fitness

ABSTRACT: Tumour DNA contains thousands of somatic single nucleotide variants (SNVs) in non-protein-coding elements, yet their functional significance remains poorly understood. Amongst the most highly mutated elements are long noncoding RNAs (lncRNAs), functional transcripts with known roles in carcinogenesis. To search for driver mutations in lncRNAs, we apply an integrative driver discovery algorithm to SNVs from 2583 primary tumours and 3527 metastases to reveal 54 potential “driver lncRNAs”. Our algorithm confirms a particularly high mutation rate in the iconic cancer lncRNA, NEAT1, which has been ascribed by recent studies to passenger effects. We directly test the functionality of NEAT1 SNVs using in cellulo mutagenesis, identifying discrete regions where mutations reproducibly increase cell proliferation in diverse cell backgrounds, both cancerous and normal. In particular, mutations in the 5’ region alter ribonucleoprotein assembly and boost the population of subnuclear paraspeckles, thus mechanistically linking mutations to cellular proliferation. We then used RNA-pull down followed by mass spectrometry to identify the protein interactor changing between the wild type and mutant form of NEAT1.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

DISEASE(S): Cancer

SUBMITTER: Manfred Heller

LAB HEAD: Rory Johnson

PROVIDER: PXD034007 | Pride | 2023-07-20

REPOSITORIES: Pride

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Publications

Tumour mutations in long noncoding RNAs enhance cell fitness.

Esposito Roberta R Lanzós Andrés A Uroda Tina T Ramnarayanan Sunandini S Büchi Isabel I Polidori Taisia T Guillen-Ramirez Hugo H Mihaljevic Ante A Merlin Bernard Mefi BM Mela Lia L Zoni Eugenio E Hovhannisyan Lusine L McCluggage Finn F Medo Matúš M Basile Giulia G Meise Dominik F DF Zwyssig Sandra S Wenger Corina C Schwarz Kyriakos K Vancura Adrienne A Bosch-Guiteras Núria N Andrades Álvaro Á Tham Ai Ming AM Roemmele Michaela M Medina Pedro P PP Ochsenbein Adrian F AF Ochsenbein Adrian F AF Riether Carsten C Kruithof-de Julio Marianna M Zimmer Yitzhak Y Medová Michaela M Stroka Deborah D Fox Archa A Johnson Rory R

Nature communications 20230608 1

Long noncoding RNAs (lncRNAs) are linked to cancer via pathogenic changes in their expression levels. Yet, it remains unclear whether lncRNAs can also impact tumour cell fitness via function-altering somatic "driver" mutations. To search for such driver-lncRNAs, we here perform a genome-wide analysis of fitness-altering single nucleotide variants (SNVs) across a cohort of 2583 primary and 3527 metastatic tumours. The resulting 54 mutated and positively-selected lncRNAs are significantly enriched ...[more]

PMID: 37291246

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Project description:Long noncoding RNAs (lncRNAs) are important regulators of chromatin; however, the mechanistic roles for many lncRNAs are poorly understood in part because their direct interactions with genomic loci and proteins are difficult to assess. We used CHART-seq to map the genomic binding sites for two highly expressed human lncRNAs, NEAT1 and MALAT1, which localize within the nucleus to paraspeckles and nuclear speckles, respectively. We show that NEAT1 and MALAT1 localize to hundreds of genomic sites in human cells, primarily over active genes. NEAT1 and MALAT1 exhibit colocalization to many of these loci, but display distinct gene body binding patterns at these sites, suggesting independent but complementary functions for these RNAs. Protein mass spectrometry analysis of CHART-enriched material (CHART-MS) identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function, in addition to unique associated proteins. Transcriptional inhibition or stimulation affects the localization of NEAT1 to active chromatin sites, implying that DNA sequence itself does not target NEAT1 to chromatin and that localization responds to cues involved in the transcription process. Paired-end CHART-seq was performed for a single replicate of each capture oligonucleotide in untreated MCF-7 cells to establish binding sites of these RNAs, for a total of 6 samples. To investigate the effects of transcriptional inhibition and E2 stimulation on the localization of these RNAs, we performed paired-end CHART-seq with each capture oligonucleotide for two biological replicates of flavopiridol- and vehicle (DMSO)-treated MCF-7 cells and for two biological replicates of E2- and vehicle (ethanol)-treated MCF-7 cells. To establish the overlap of NEAT1 and MALAT1 binding sites with a known component of paraspeckles (NEAT1-containing subnuclear body), we performed paired-end ChIP-seq for the paraspeckle component PSF in MCF-7 cells, as well as a single-end biological replicate.

Project description:Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-ACnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays we show that Top3 unlike Top1 and Top2 is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-ACnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-ACnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-ACnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology which in turn affects the dynamics of CENP-ACnp1 nucleosomes. For transcription: Total RNA from top3-105 mutant and WT control cells after 8 hours at 36C in biological duplicates. For Top3-myc chromatin immunoprecipitation: DNA immunoprecipitated with mouse anti-Myc using chromatin extracts from cells expressing Top3-Myc from the endogenous locus at 30C in biological duplicates normalized to input DNA from wild type cells at 30C in biological duplicates. For CENP-A/Cnp1 chromatin immunoprecipitation: DNA immunoprecipitated with anti-Cnp1 serum using chromatin extracts from top3-105 mutant and wild type control cells after 8 hours at 36C in in biological duplicates normalized to input DNA from each strain.

Dataset Information

Tumour mutations in long noncoding RNAs enhance cell fitness

Publications

Tumour mutations in long noncoding RNAs enhance cell fitness.

Similar Datasets

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