Many recent studies established a potential role for p53 in translational control of many transcripts through the modulation of the expression of several microRNAs, RNA binding proteins (RBPs), translation factors, or of ribosome biogenesis factors. To establish the relevance of translation control in p53-dependent apoptosis our group recently compared by polysome profiling two cell lines, SJSA1 and HCT116, undergoing respectively p53-dependent apoptosis and cell cycle arrest in response to the treatment with Nutlin-3, a selective activator of p53. Examining the markedly different, treatment-induced translation landscapes, the RNA helicase DHX30 was identified as a protein able to bind only in HCT116 cells to a 3’-UTR sequence motif, labeled as CGPD-motif. The motif had been identified as highly over-represented among translationally enhanced mRNAs in nutlin-treated SJSA1 cells, among which apoptosis effectors were enriched. Since the binding of DHX30 to the CGPD-motif transcripts is observed in cell cycle arresting HCT116, where those mRNA are not translationally enhanced, we reasoned that DHX30 could be part of an RBP complex responsible for reduced translation of specific pro-apoptotic mRNAs. To pursue this hypothesis, HCT116-shDHX30 clones were obtained and I studied the functions of this relatively unknown RNA helicase in this and other cell models. A translatome analysis by RNA-seq of HCT116-shDHX30 cells showed higher activation of ribosome biogenesis pathways, according to GSEA. Consistently, eCLIP data from ENCODE indicate that DHX30 can bind to most ribosomal protein transcripts, and those showed higher relative translation efficiency in HCT116-shDHX30 cells. In fact, the cells exhibited increased rRNA synthesis and higher global translation as well as a large expansion of the number of transcripts showing higher polysome-association in response to Nutlin. The DHX30 gene features two promoters, one of which can produce a transcript containing a mitochondrial localization signal. I confirmed that DHX30 has both a cytoplasmic and a mitochondrial localization by both cell fractionation and immunofluorescence. HCT116_shDHX30 cells have reduced mitochondrial metabolism, based on oxygen consumption rates but do not show evidence of compensatory glycolytic activity. Impaired mitochondrial functions could be related also to decreased expression of mitochondrial oxidative phosphorylation (OXPHOS) components as well as reduced expression of nuclear encoded mitoribosome proteins. Based on RIP assays DHX30 appears to target directly mitoribosome transcripts. Depletion of DHX30 showed reduced proliferation in various assays both in 2D and 3D culture conditions and sensitized cells to the treatment with the mitochondrial uncoupler FCCP. A similar role was observed also on mitochondrially encoded OXPHOS compomenents as well as mitoribosome components in MCF7 breast cancer cell line silenced for DHX30 with a consistent reduction in cell proliferation. In contrast, U2OS osteosarcoma cells did not show a change in the expression in mitochondrial components as well as an impact on proliferation after transient DHX30 silencing. Hence, it appears that DHX30 can exert a broad general control on cell metabolism and translation as well as a cis-element mediated control of the translation of specific mRNAs impacting both on cell fitness and apoptosis.
Exploring DHX30, an RNA helicase that coordinates cytoplasmic translation and mitochondrial function contributing to cancer cell survival / Bosco, Bartolomeo. - (2020 Nov 03), pp. 1-125.
|Titolo:||Exploring DHX30, an RNA helicase that coordinates cytoplasmic translation and mitochondrial function contributing to cancer cell survival|
|Anno di pubblicazione:||2020-11-03|
|Struttura:||Dipartimento di Biologia Cellulare, Computazionale e Integrata - CIBIO|
|Corso di dottorato:||Biomolecular Sciences|
|Tesi in cotutela:||no|
|Digital Object Identifier (DOI):||10.15168/11572_277809|
|Appare nelle tipologie:||08.1 Tesi di dottorato (Doctoral Thesis)|
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