Abstract
Diversification at the transcriptome 3′end is an important and evolutionarily conserved layer of gene regulation associated with differentiation and dedifferentiation processes. Here, we identify extensive transcriptome 3′end-alterations in neuroblastoma, a tumour entity with a paucity of recurrent somatic mutations and an unusually high frequency of spontaneous regression. Utilising extensive RNAi-screening we reveal the landscape and drivers of transcriptome 3′end-diversification, discovering PCF11 as critical regulator, directing alternative polyadenylation (APA) of hundreds of transcripts including a differentiation RNA-operon. PCF11 shapes inputs converging on WNT-signalling, and governs cell cycle, proliferation, apoptosis and neurodifferentiation. Postnatal PCF11 down-regulation induces a neurodifferentiation program, and low-level PCF11 in neuroblastoma associates with favourable outcome and spontaneous tumour regression. Our findings document a critical role for APA in tumorigenesis and describe a novel mechanism for cell fate reprogramming in neuroblastoma with potentially important clinical implications. We provide an interactive data repository of transcriptome-wide APA covering > 170 RNAis, and an APA-network map with regulatory hubs.
Highlights
Diversification at the transcriptome 3′end is an important and evolutionarily conserved layer of gene regulation associated with differentiation and dedifferentiation processes
By employing a tailored approach based on RNA 3′ region extraction and deep sequencing[15], we observed a significant change of transcripts with alternative 3′ ends upon BE(2)-C differentiation with a trend towards shorter transcript isoforms, predominantly affecting 3′ untranslated regions (UTRs) (Fig. 1c)
We further identified a substantial fraction of APAaffected genes (n = 74) that are shared in the clinical cohort with those regulated upon PCF11 depletion in BE(2)-C cells
Summary
Diversification at the transcriptome 3′end is an important and evolutionarily conserved layer of gene regulation associated with differentiation and dedifferentiation processes. The clinical presentation is heterogeneous, ranging from an asymptomatic tumour disease to a critical illness as a result of local invasion, or as widely disseminated disease This tumour entity is generally characterised by a lack of recurrent somatic mutations, and exhibits one of the highest proportions of spontaneous and complete regression of all human cancers by as yet unknown mechanisms[1,2]. By combining a genome-wide high-throughput analysis with a comprehensive RNAi screening targeting more than 170 potential components involved in the definition of RNA 3′ ends, we delineate the dynamic landscape of and explore mechanisms influencing transcriptome 3′end diversification in this tumour entity. By generating and applying an inducible short hairpin RNA (shRNA) mouse model targeting PCF11 complemented by studies of neuroblastoma patient samples, we discover an unexpected critical role for PCF11dependent APA regulation in neuronal differentiation with potentially important implications for spontaneous tumour regression
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