Reduction of toxic RNAs in myotonic dystrophies type 1 and type 2 by the RNA helicase p68/DDX5.
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ABSTRACT:
SUBMITTER: Jones K
PROVIDER: S-EPMC4491770 | biostudies-literature | 2015 Jun
REPOSITORIES: biostudies-literature
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Project description:DDX5 (p68) is a well-known multifunctional DEAD-box RNA helicase and a transcription cofactor. Since its initial discovery more than three decades ago, DDX5 is gradually recognized as a potential biomarker and target for the treatment of various cancer types. Studies over the years significantly expanded our understanding of the functional diversity of DDX5 in various cancer types and extended our knowledge of its Mechanism of Action (MOA). This provides a rationale for the development of novel cancer therapeutics by using DDX5 as a biomarker and a therapeutic target. However, while most of the published studies have found DDX5 to be an oncogenic target and a cancer treatment-resistant biomarker, a few studies have reported that in certain scenarios, DDX5 may act as a tumor suppressor. After careful review of all the available relevant studies in the literature, we found that the multiple functions of DDX5 make it both a superior independent oncogenic biomarker and target for targeted cancer therapy. In this article, we will summarize the relevant studies on DDX5 in literature with a careful analysis and discussion of any inconsistencies encountered, and then provide our conclusions with respect to understanding the MOA of FL118, a novel small molecule. We hope that such a review will stimulate further discussion on this topic and assist in developing better strategies to treat cancer by using DDX5 as both an oncogenic biomarker and therapeutic target.
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Project description:Non-sense-mediated mRNA decay (NMD) is a mechanism of translation-dependent mRNA surveillance in eukaryotes: it degrades mRNAs with premature termination codons (PTCs) and contributes to cellular homeostasis by downregulating a number of physiologically important mRNAs. In the NMD pathway, Upf proteins, a set of conserved factors of which Upf1 is the central regulator, recruit decay enzymes to promote RNA cleavage. In mammals, the degradation of PTC-containing mRNAs is triggered by the exon-junction complex (EJC) through binding of its constituents Upf2 and Upf3 to Upf1. The complex formed eventually induces translational repression and recruitment of decay enzymes. Mechanisms by which physiological mRNAs are targeted by the NMD machinery in the absence of an EJC have been described but still are discussed controversially. Here, we report that the DEAD box proteins Ddx5/p68 and its paralog Ddx17/p72 also bind the Upf complex by physical interaction with Upf3, thereby interfering with the binding of EJC. By activating the NMD machinery, Ddx5 is shown to regulate the expression of its own, Ddx17 and Smg5 mRNAs. For NMD triggering, the adenosine triphosphate-binding activity of Ddx5 and the 3'-untranslated region of substrate mRNAs are essential.
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Project description:Myotonic Dystrophy type I (DM1) is caused by an abnormal expansion of CTG triplets in the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene, leading to the aggregation of the mutant transcript in nuclear RNA foci. The expanded mutant transcript promotes the sequestration of the MBNL1 splicing factor, resulting in the misregulation of a subset of alternative splicing events. In this study, we identify the DEAD-box RNA helicase p68 (DDX5) in complexes assembled onto in vitro-transcribed CUG repeats. We showed that p68 colocalized with RNA foci in cells expressing the 3'UTR of the DMPK gene containing expanded CTG repeats. We found that p68 increased MBNL1 binding onto pathological repeats and the stem-loop structure regulatory element within the cardiac Troponin T (TNNT2) pre-mRNA, splicing of which is misregulated in DM1. Mutations in the helicase core of p68 prevented both the stimulatory effect of the protein on MBNL1 binding and the colocalization of p68 with CUG repeats, suggesting that remodeling of RNA secondary structure by p68 facilitates MBNL1 binding. We also found that the competence of p68 for regulating TNNT2 exon 5 inclusion depended on the integrity of MBNL1 binding sites. We propose that p68 acts as a modifier of MBNL1 activity on splicing targets and pathogenic RNA.
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Project description:The DEAD-box RNA helicase p68 (DDX5) plays important roles in several cellular processes, including transcription, pre-mRNA processing, and microRNA (miRNA) processing. p68 expression is growth and developmentally regulated, and alterations in p68 expression and/or function have been implicated in tumor development. The p68 gene encodes an evolutionarily conserved, alternatively spliced, intron the function of which has to date remained unclear. Although the intron-containing p68 RNA does not appear to yield an alternative p68 protein, it is differentially expressed in cell lines and tissues, indicating regulation of expression. Here we show that the p68 conserved intron encodes a novel putative miRNA, suggesting a previously unknown possible regulatory function for the p68 intron. We show that this miRNA (referred to as p68 miRNA) is processed from the intron via the canonical miRNA-processing pathway and that it associates with the Argonaute protein Ago2. Finally we show that the p68 miRNA suppresses an mRNA bearing complementary target sequences, suggesting that it is functional. These findings suggest a novel mechanism by which alterations in p68 expression may impact on the cell.
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Project description:BackgroundH-Ras pre-mRNA undergoes an alternative splicing process to render two proteins, namely p21 H-Ras and p19 H-Ras, due to either the exclusion or inclusion of the alternative intron D exon (IDX), respectively. p68 RNA helicase (p68) is known to reduce IDX inclusion.Principal findingsHere we show that p68 unwinds the stem-loop IDX-rasISS1 structure and prevents binding of hnRNP H to IDX-rasISS1. We also found that p68 alters the dynamic localization of SC35, a splicing factor that promotes IDX inclusion. The knockdown of hnRNP A1, FUS/TLS and hnRNP H resulted in upregulation of the expression of the gene encoding the SC35-binding protein, SFRS2IP. Finally, FUS/TLS was observed to upregulate p19 expression and to stimulate IDX inclusion, and in vivo RNAi-mediated depletion of hnRNP H decreased p19 H-Ras abundance.SignificanceTaken together, p68 is shown to be an essential player in the regulation of H-Ras expression as well as in a vital transduction signal pathway tied to cell proliferation and many cancer processes.
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Project description:It is widely accepted that pre-mRNA maturation, including splicing, is tightly coupled to both transcription and mRNA export, but factors linking the three processes are less understood. By analysing the estrogen-regulated expression of the c-fos mRNA that is processed during transcription, we show that the ddx5 RNA helicase, is required throughout the major nuclear steps of the expression of the c-fos gene, from transcription to mRNA export. Indeed, ddx5, whose recruitment on the c-fos gene was increased upon estrogen treatment, was required for the full transcriptional activation of the c-fos gene. In addition, ddx5 was required for c-fos co-transcriptional RNA splicing. When splicing occurred post-transcriptionally in the absence of ddx5, the c-fos mRNA was poorly exported into the cytosol because of inefficient recruitment of the TAP mRNA export receptor. Finally, ddx5 was present in the c-fos messenger ribonucleoprotein together with mRNA export factors, which further supports that ddx5 is a key operator in the c-fos 'mRNA factory'.
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Project description:For some neurological disorders, disease is primarily RNA mediated due to expression of non-coding microsatellite expansion RNAs (RNA(exp)). Toxicity is thought to result from enhanced binding of proteins to these expansions and depletion from their normal cellular targets. However, experimental evidence for this sequestration model is lacking. Here, we use HITS-CLIP and pre-mRNA processing analysis of human control versus myotonic dystrophy (DM) brains to provide compelling evidence for this RNA toxicity model. MBNL2 binds directly to DM repeat expansions in the brain, resulting in depletion from its normal RNA targets with downstream effects on alternative splicing and polyadenylation. Similar RNA processing defects were detected in Mbnl compound-knockout mice, highlighted by dysregulation of Mapt splicing and fetal tau isoform expression in adults. These results demonstrate that MBNL proteins are directly sequestered by RNA(exp) in the DM brain and introduce a powerful experimental tool to evaluate RNA-mediated toxicity in other expansion diseases.
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Project description:The RNA helicase p68 (DDX5) is an established co-activator of the p53 tumour suppressor that itself has a pivotal role in orchestrating the cellular response to DNA damage. Although several factors influence the biological outcome of p53 activation, the mechanisms governing the choice between cell-cycle arrest and apoptosis remain to be elucidated. In the present study, we show that, while p68 is critical for p53-mediated transactivation of the cell-cycle arrest gene p21(WAF1/CIP1), it is dispensable for induction of several pro-apoptotic genes in response to DNA damage. Moreover, p68 depletion results in a striking inhibition of recruitment of p53 and RNA Pol II to the p21 promoter but not to the Bax or PUMA promoters, providing an explanation for the selective effect on p21 induction. Importantly, these findings are mirrored in a novel inducible p68 knockout mouse model in which p68 depletion results in a selective inhibition of p21 induction in several tissues. Moreover, in the bone marrow, p68 depletion results in an increased sensitivity to ?-irradiation, consistent with an increased level of apoptosis. These data highlight a novel function of p68 as a modulator of the decision between p53-mediated growth arrest and apoptosis in vitro and in vivo.
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Project description:A working hypothesis for the pathogenesis of myotonic dystrophy type 1 (DM1) involves the aberrant sequestration of an alternative splicing regulator, MBNL1, by expanded CUG repeats, r(CUG)(exp). It has been suggested that a reversal of the myotonia and potentially other symptoms of the DM1 disease can be achieved by inhibiting the toxic MBNL1-r(CUG)(exp) interaction. Using rational design, we discovered an RNA-groove binding inhibitor (ligand 3) that contains two triaminotriazine units connected by a bisamidinium linker. Ligand 3 binds r(CUG)12 with a low micromolar affinity (K(d) = 8 ± 2 ?M) and disrupts the MBNL1-r(CUG)12 interaction in vitro (K(i) = 8 ± 2 ?M). In addition, ligand 3 is cell and nucleus permeable, exhibits negligible toxicity to mammalian cells, dissolves MBNL1-r(CUG)(exp) ribonuclear foci, and restores misregulated splicing of IR and cTNT in a DM1 cell culture model. Importantly, suppression of r(CUG)(exp) RNA-induced toxicity in a DM1 Drosophila model was observed after treatment with ligand 3. These results suggest ligand 3 as a lead for the treatment of DM1.
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Project description:Myotonic dystrophy type 2 (DM2) is caused by an expansion of CCTG repeats in the zinc-finger protein gene (ZNF9). Transcribed CCUG repeats sequester muscleblind-like protein 1 (MBNL1), an important alternative splicing regulator, preventing its normal function, leading to the disease phenotype. We describe a series of ligands that disrupt the MBNL1-r(CCUG)n interaction as potential lead agents for developing DM2 therapeutics. A previously reported triaminopyrimidine-acridine conjugate was a moderate inhibitor in vitro, however it proved to be poorly water-soluble and not cell-permeable. To improve its therapeutic potential, the new set of ligands maintained the key triaminopyrimidine recognition unit but replaced the acridine intercalator with a bisamidinium groove binder. The optimized ligands exhibit low micromolar inhibition potency to MBNL1-r(CCUG)8. Importantly, the ligands are the first to show the ability to disrupt the MBNL1-r(CCUG)n foci in DM2 model cell culture and exhibit low cytotoxicity.
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