Multiplexed functional genomic analysis of somatic 5' untranslated region mutations across the spectrum of human prostate cancer (RNASeq)
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE171729 | GEO | 2021/05/17
REPOSITORIES: GEO
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Project description:The functional consequences of cancer patient-derived genetic variants within the 5’ untranslated regions (UTRs) on a genome-wide scale and their effects on mRNA transcript and translation levels are poorly understood. To systematically interrogate the mutational landscape of 5’ UTRs across cancer patients with localized to metastatic disease, we analyzed the genomes of 226 prostate cancer patients and observed thousands of mutations, many of which impact known cis-regulatory elements. We developed a high-throughput multi-layer massively parallel sequencing-based method called PLUMAGE (Pooled full-length UTR Multiplex Assay on Gene Expression) to simultaneously quantify the effects of 545 5’ UTR somatic mutations across recurrently mutated or cancer-related genes from our patient cohort. Our method enabled unprecedented insights into how 5’ UTR mutations can control multiple levels of gene expression simultaneously. In particular, we identified 190 mutations that significantly altered 5’ UTR function, either by creating new DNA binding elements, disrupting known translation regulatory motifs, or simultaneously impacting both transcript levels and mRNA translation. Furthermore, we also determined that 5’ UTR mutations to the MAP kinase signaling pathway are significantly associated with early metastasis. This study is the first to comprehensively interrogate the functional 5’ UTR mutational landscape of a human cancer revealing the importance of untranslated regions in regulating multiple levels of oncogenic gene expression and provides a high-throughput functional genomics solution applicable to many genetically driven diseases.
2021-05-17 | GSE149487 | GEO
Project description:The functional consequences of cancer patient-derived genetic variants within the 5’ untranslated regions (UTRs) on a genome-wide scale and their effects on mRNA transcript and translation levels are poorly understood. To systematically interrogate the mutational landscape of 5’ UTRs across cancer patients with localized to metastatic disease, we analyzed the genomes of 226 prostate cancer patients and observed thousands of mutations, many of which impact known cis-regulatory elements. We developed a high-throughput multi-layer massively parallel sequencing-based method called PLUMAGE (Pooled full-length UTR Multiplex Assay on Gene Expression) to simultaneously quantify the effects of 545 5’ UTR somatic mutations across recurrently mutated or cancer-related genes from our patient cohort. Our method enabled unprecedented insights into how 5’ UTR mutations can control multiple levels of gene expression simultaneously. In particular, we identified 190 mutations that significantly altered 5’ UTR function, either by creating new DNA binding elements, disrupting known translation regulatory motifs, or simultaneously impacting both transcript levels and mRNA translation. Furthermore, we also determined that 5’ UTR mutations to the MAP kinase signaling pathway are significantly associated with early metastasis. This study is the first to comprehensively interrogate the functional 5’ UTR mutational landscape of a human cancer revealing the importance of untranslated regions in regulating multiple levels of oncogenic gene expression and provides a high-throughput functional genomics solution applicable to many genetically driven diseases.
2021-05-17 | GSE130465 | GEO
Project description:The functional consequences of cancer patient-derived genetic variants within the 5’ untranslated regions (UTRs) on a genome-wide scale and their effects on mRNA transcript and translation levels are poorly understood. To systematically interrogate the mutational landscape of 5’ UTRs across cancer patients with localized to metastatic disease, we analyzed the genomes of 226 prostate cancer patients and observed thousands of mutations, many of which impact known cis-regulatory elements. We developed a high-throughput multi-layer massively parallel sequencing-based method called PLUMAGE (Pooled full-length UTR Multiplex Assay on Gene Expression) to simultaneously quantify the effects of 545 5’ UTR somatic mutations across recurrently mutated or cancer-related genes from our patient cohort. Our method enabled unprecedented insights into how 5’ UTR mutations can control multiple levels of gene expression simultaneously. In particular, we identified 190 mutations that significantly altered 5’ UTR function, either by creating new DNA binding elements, disrupting known translation regulatory motifs, or simultaneously impacting both transcript levels and mRNA translation. Furthermore, we also determined that 5’ UTR mutations to the MAP kinase signaling pathway are significantly associated with early metastasis. This study is the first to comprehensively interrogate the functional 5’ UTR mutational landscape of a human cancer revealing the importance of untranslated regions in regulating multiple levels of oncogenic gene expression and provides a high-throughput functional genomics solution applicable to many genetically driven diseases.
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Project description:Metastatic, castration-resistant prostate cancer (mCRPC) is an advanced form of prostate cancer with a high mortality rate due to a current lack of treatment options. While much is already known about how mutations in protein-coding sequences across the genome affect prostate cancer, somatic mutations occurring in the 3’ untranslated regions (3’UTRs) of genes are largely unstudied. The 3’UTR is a genomic region that controls post-transcriptional gene expression through its recruitment of trans-acting factors such as RNA-binding proteins (RBPs) and microRNAs (miRNAs), which themselves are known to be oncogenes and tumor suppressors in many cases. To better understand the role of 3’UTR mutations across prostate cancer, we have created a database of 3’UTR somatic mutations in 185 patients with mCRPC, discovering 14,497 single-nucleotide mutations throughout the 3’UTRome. In order to functionally assay these variants, we have developed a novel pair of massively parallel reporter assays (MPRA) able to determine the effect of thousands of patient somatic mutations on post-transcriptional gene expression. In this two-pronged approach, we are able to measure whether each of 6,892 mutations found in recurrently mutated 3’UTRs affect mRNA stability, steady-state transcript level, and translation efficiency. This deep functional assessment of thousands of 3’UTR mutations allows us to uncover patterns in mutation functionality, including their association with RNA motifs and sequence conservation. Investigation into how the resultant gene expression changes from 3’UTR mutations affect prostate cancer pathogenesis, such as cancer growth or response to treatment, is also underway. This work represents an unprecedented view of the extent to which disease-relevant 3’UTR mutations affect mRNA stability, translation efficiency, and cancer phenotypes, expanding the boundaries of functional cancer genomics and potentially uncovering novel therapeutic targets in previously unexplored regulatory regions.
2023-06-30 | GSE217530 | GEO
Project description:Metastatic, castration-resistant prostate cancer (mCRPC) is an advanced form of prostate cancer with a high mortality rate due to a current lack of treatment options. While much is already known about how mutations in protein-coding sequences across the genome affect prostate cancer, somatic mutations occurring in the 3’ untranslated regions (3’UTRs) of genes are largely unstudied. The 3’UTR is a genomic region that controls post-transcriptional gene expression through its recruitment of trans-acting factors such as RNA-binding proteins (RBPs) and microRNAs (miRNAs), which themselves are known to be oncogenes and tumor suppressors in many cases. To better understand the role of 3’UTR mutations across prostate cancer, we have created a database of 3’UTR somatic mutations in 185 patients with mCRPC, discovering 14,497 single-nucleotide mutations throughout the 3’UTRome. In order to functionally assay these variants, we have developed a novel pair of massively parallel reporter assays (MPRA) able to determine the effect of thousands of patient somatic mutations on post-transcriptional gene expression. In this two-pronged approach, we are able to measure whether each of 6,892 mutations found in recurrently mutated 3’UTRs affect mRNA stability, steady-state transcript level, and translation efficiency. This deep functional assessment of thousands of 3’UTR mutations allows us to uncover patterns in mutation functionality, including their association with RNA motifs and sequence conservation. Investigation into how the resultant gene expression changes from 3’UTR mutations affect prostate cancer pathogenesis, such as cancer growth or response to treatment, is also underway. This work represents an unprecedented view of the extent to which disease-relevant 3’UTR mutations affect mRNA stability, translation efficiency, and cancer phenotypes, expanding the boundaries of functional cancer genomics and potentially uncovering novel therapeutic targets in previously unexplored regulatory regions.
2023-06-30 | GSE200302 | GEO
Project description:Metastatic, castration-resistant prostate cancer (mCRPC) is an advanced form of prostate cancer with a high mortality rate due to a current lack of treatment options. While much is already known about how mutations in protein-coding sequences across the genome affect prostate cancer, somatic mutations occurring in the 3’ untranslated regions (3’UTRs) of genes are largely unstudied. The 3’UTR is a genomic region that controls post-transcriptional gene expression through its recruitment of trans-acting factors such as RNA-binding proteins (RBPs) and microRNAs (miRNAs), which themselves are known to be oncogenes and tumor suppressors in many cases. To better understand the role of 3’UTR mutations across prostate cancer, we have created a database of 3’UTR somatic mutations in 185 patients with mCRPC, discovering 14,497 single-nucleotide mutations throughout the 3’UTRome. In order to functionally assay these variants, we have developed a novel pair of massively parallel reporter assays (MPRA) able to determine the effect of thousands of patient somatic mutations on post-transcriptional gene expression. In this two-pronged approach, we are able to measure whether each of 6,892 mutations found in recurrently mutated 3’UTRs affect mRNA stability, steady-state transcript level, and translation efficiency. This deep functional assessment of thousands of 3’UTR mutations allows us to uncover patterns in mutation functionality, including their association with RNA motifs and sequence conservation. Investigation into how the resultant gene expression changes from 3’UTR mutations affect prostate cancer pathogenesis, such as cancer growth or response to treatment, is also underway. This work represents an unprecedented view of the extent to which disease-relevant 3’UTR mutations affect mRNA stability, translation efficiency, and cancer phenotypes, expanding the boundaries of functional cancer genomics and potentially uncovering novel therapeutic targets in previously unexplored regulatory regions.
2023-06-30 | GSE200303 | GEO
Project description:De novo mutations cause a variety of neurodevelopmental disorders including autism. Recent whole genome sequencing has identified hundreds of mutations in untranslated regions (UTRs) of genes from individuals with autism, but it is impossible to predict from sequence alone which are functional, and thus might be causal. Therefore, we developed a high throughput assay to screen the consequence over 1,000 variants from 5'UTRs mutations on transcript abundance and translation efficiency. This assay successfully enriched for elements that alter reporter translation, identifying over 100 potentially functional mutations. Studies in patient-derived cell lines further confirmed these mutations alter protein production in individuals in autism, including for multiple genes known to cause of syndromic forms autism, suggesting a diagnosis for these individual patients. Since UTR function varies by cell type, we further optimized this high throughput assay to enable assessment of mutations in neurons of the living brain. Neurons demonstrate profoundly different principles of regulation by 5'UTRs, consistent with more robust mechanism for reducing impact of 5'UTR RNA structure. Overall our results highlight a new approach for assessing the impact of 5’UTR across cell types and suggest some cases of neurodevelopmental disorder may be caused by such variants.
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Project description:The proper subcellular localization of RNAs and local translational regulation is crucial in highly compartmentalized cells, such as neurons. RNA localization is mediated by specific cis-regulatory elements usually found in mRNA 3'UTRs. Therefore, processes that generate alternative 3'UTRs – alternative splicing and polyadenylation – have the potential to diversify mRNA localization patterns in neurons. Here, we performed mapping of alternative 3'UTRs in neurites and soma isolated from mESC-derived neurons. Our analysis identified 593 genes with differentially localized 3'UTR isoforms. In particular, we have shown that two isoforms of Cdc42 gene with distinct functions in neuronal polarity are differentially localized between neurites and soma of mESC-derived and mouse primary cortical neurons, at both mRNA and protein level. Using reporter assays and 3'UTR swapping experiments, we have identified the role of alternative 3’UTRs and mRNA transport in differential localization of alternative CDC42 protein isoforms. Moreover, we used SILAC to identify isoform-specific Cdc42 3'UTR-bound proteome with potential role in Cdc42 localization and translation. Our analysis points to usage of alternative 3'UTR isoforms as a novel mechanism to provide for differential localization of functionally diverse alternative protein isoforms.
2018-12-07 | PXD011518 | Pride
Project description:mTOR regulates mRNA translation. Whereas ribosome-profiling suggested that mTOR exclusively stimulates translation of TOP (containing a 5â-terminal oligopyrimidine [5âTOP] motif) and TOP-like mRNAs, polysome-profiling implied that mTOR also modulates translation of non-TOP mRNAs. We show that ribosome-, but not polysome-profiling, is biased towards identification of TOP mRNAs as differentially translated while obscuring detection of changes in non-TOP mRNA translation. Transcription start site profiling by Nano-Cap Analysis of Gene Expression (nanoCAGE) revealed that many mTOR-sensitive mRNAs do not have 5âTOP motifs. Moreover, nanoCAGE showed that 5â UTR features distinguish two functionally and translationally distinct subsets of mTOR-sensitive mRNAs: i) those with short 5â UTRs enriched for mitochondrial functions such as respiration, that are translated in an eIF4E, but not eIF4A1-dependent manner and ii) mRNAs encoding proliferation- and survival-promoting proteins, that harbor long 5â UTRs, and require both eIF4E and eIF4A1 for their efficient translation. Selective inhibition of translation of mRNAs harboring long 5â UTRs via suppression of eIF4A leads to uncoupling of expression of proteins involved in respiration (e.g. ATP5O) from those protecting mitochondrial integrity (e.g. BCL-2) ultimately resulting in apoptosis. Conversely, simultaneous translational downregulation of both long and short 5â UTR mRNAs by mTOR inhibitors results in suppression of mitochondrial respiration and predominantly cytostatic effects. Therefore, 5â UTR features define differential modes of translation of functionally distinct mTOR-sensitive mRNAs, which explains discrepancies between the effects of mTOR and eIF4A inhibitors on neoplastic cells. Determination of 5'UTR lengths using nanoCAGE in MCF7 cells
2016-03-14 | E-GEOD-77033 | biostudies-arrayexpress
Project description:The 3' untranslated regions (3' UTRs) of mRNAs play important roles in the regulation of mRNA localization, translation, and stability. Alternative cleavage and polyadenylation (APA) generates mRNAs with different 3' UTRs, but the involvement of this process in stress response has not been clarified. Here, we report that a subset of stress-related genes exhibits 3' UTR extensions of their mRNAs during dehydration stress. These extended 3' UTRs have characteristics of long noncoding RNAs and likely do not interact with miRNAs. Functional studies using T-DNA insertion mutants reveal that they can function as antisense transcripts to repress expression levels of sense genes from the opposite strand, or can activate the transcription of downstream genes from the same strand. Poly(A) signal analysis reveal that mRNAs with 3' UTR extensions have weaker poly(A) signals than mRNAs without 3' UTR extensions. Finally, we show that their biogenesis is partially dependent on a trans-acting factor FPA. Taken together, we report that dehydration stress could induce mRNA 3' UTR extensions, and elucidate a novel function for these stress-induced 3' UTR extensions as long noncoding RNAs in the regulation of their neighboring genes.
2017-04-05 | GSE74864 | GEO