Retraction Note: DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions.

Change History: This Article has been retracted; see accompanying Retraction. Corrected online 20 January: In this Article, author Frank Rigo was incorrectly listed with a middle initial; this has been corrected in the online versions of the paper.

DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions.

T helper 17 (TH17) lymphocytes protect mucosal barriers from infections, but also contribute to multiple chronic inflammatory diseases. Their differentiation is controlled by RORγt, a ligand-regulated nuclear receptor. Here we identify the RNA helicase DEAD-box protein 5 (DDX5) as a RORγt partner that coordinates transcription of selective TH17 genes, and is required for TH17-mediated inflammatory pathologies. Surprisingly, the ability of DDX5 to interact with RORγt and coactivate its targets depends on intrinsic RNA helicase activity and binding of a conserved nuclear long noncoding RNA (lncRNA), Rmrp, which is mutated in patients with cartilage-hair hypoplasia. A targeted Rmrp gene mutation in mice, corresponding to a gene mutation in cartilage-hair hypoplasia patients, altered lncRNA chromatin occupancy, and reduced the DDX5-RORγt interaction and RORγt target gene transcription. Elucidation of the link between Rmrp and the DDX5-RORγt complex reveals a role for RNA helicases and lncRNAs in tissue-specific transcriptional regulation, and provides new opportunities for therapeutic intervention in TH17-dependent diseases.

LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner.

Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53.

The miRNA biogenesis factors, p72/DDX17 and KHSRP regulate the protein level of Ago2 in human cells.

MicroRNAs (miRNAs) are short (21-23nt long) RNAs that post-transcriptionally regulate gene expression in plants and animals. They are key regulators in all biological processes. In mammalian cells miRNAs are loaded into one of the four members of the Argonaute (Ago) protein family to form the RNA-induced silencing complex (RISC). RISCs inhibit the translation of mRNAs that share sequence complementarity with their loaded miRNAs. miRNA processing and miRNA-mediated gene regulation are highly regulated processes and involve many RNA-binding proteins as auxiliary factors. Here we show that the two RNA-binding proteins, p72 and KHSRP, both with known roles in promoting miRNA biogenesis, regulate the protein level of human Ago2 in transformed human cells. We determined that p72 and KHSRP influence Ago2 stability by regulating miRNA levels in the cell and that loss of p72/KHSRP results in a decrease of unloaded Ago2.

The DEAD box proteins DDX5 (p68) and DDX17 (p72): multi-tasking transcriptional regulators.

Members of the DEAD box family of RNA helicases, which are characterised by the presence of twelve conserved motifs (including the signature D-E-A-D motif) within a structurally conserved 'helicase' core, are involved in all aspects of RNA metabolism. Apart from unwinding RNA duplexes, which established these proteins as RNA helicases, DEAD box proteins have been shown to also catalyse RNA annealing and to displace proteins from RNA. DEAD box proteins generally act as components of large multi-protein complexes and it is thought that interactions, via their divergent N- and C-terminal extensions, with other factors in the complexes may be responsible for the many different functions attributed to these proteins. In addition to their established crucial roles in the manipulation of RNA structure, it is becoming increasingly clear that several members of the DEAD box family act as regulators of transcription. In this review I shall focus on DDX5 (p68) and the highly related DDX17 (p72), two proteins for which there is a large body of evidence demonstrating that they function in transcriptional regulation. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life.

Looking back on the birth of DEAD-box RNA helicases.

DEAD-box proteins represent the largest family of RNA helicases, present in all three kingdoms of life. They are involved in a variety of processes involving RNA metabolism and in some instances also in processes that use guide RNAs. Since their first descriptions in the late 1980s, the perception of their molecular activities has dramatically changed. At the time when only eight proteins with 9 conserved motifs constituted the DEAD-box protein family, it was the biochemical characterization of mammalian eIF4A that first suggested a local unwinding activity. This was confirmed in vitro using partially double stranded RNA substrates with the unexpected result of a bidirectional unwinding activity. A real change of paradigm from the classical helicase activity to localized RNA unwinding occurred with the publication of the vasa•RNA structure with a bend in the RNA substrate and the insightful work from several laboratories demonstrating local unwinding without translocation. Finally, elegant work on the exon-junction complex revealed how DEAD-box proteins can bind to RNA to serve as clamps to function as nucleation centers to form RNP complexes. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life.

An evolutionarily conserved, alternatively spliced, intron in the p68/DDX5 DEAD-box RNA helicase gene encodes a novel miRNA.

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.

DEAD box RNA helicase functions in cancer.

Members of the DEAD box family of RNA helicases are known to be involved in most cellular processes that require manipulation of RNA structure and, in many cases, exhibit other functions in addition to their established ATP-dependent RNA helicase activities. They thus play critical roles in cellular metabolism and in many cases have been implicated in cellular proliferation and/or neoplastic transformation. These proteins generally act as components of multi-protein complexes; therefore their precise role is likely to be influenced by their interacting partners and to be highly context-dependent. This may also provide an explanation for the sometimes conflicting reports suggesting that DEAD box proteins have both pro- and anti-proliferative roles in cancer.

The RNA helicases p68/p72 and the noncoding RNA SRA are coregulators of MyoD and skeletal muscle differentiation.

MyoD regulates skeletal myogenesis. Since proteins associated with MyoD exert regulatory functions, their identification is expected to contribute important insights into the mechanisms governing gene expression in skeletal muscle. We have found that the RNA helicases p68/p72 are MyoD-associated proteins and that the noncoding RNA SRA also immunoprecipitates with MyoD. In vitro and in vivo experiments indicated that both p68/p72 and SRA are coactivators of MyoD. RNA interference toward either p68/p72 or SRA prevented proper activation of muscle gene expression and cell differentiation. Unexpectedly, reducing the levels of p68/p72 proteins impaired recruitment of the TATA binding protein TBP; RNA polymerase II; and the catalytic subunit of the ATPase SWI/SNF complex, Brg-1, and hindered chromatin remodeling. These findings reveal that p68/p72 play a critical role in promoting the assembly of proteins required for the formation of the transcription initiation complex and chromatin remodeling.

RNA helicases p68 and p72: multifunctional proteins with important implications for cancer development.

The DEAD box RNA helicases p68 (DDX5) and p72 (DDX17) play important roles in multiple cellular processes that are commonly dysregulated in cancers, including transcription, pre-mRNA processing/alternative splicing and miRNA processing. Although p68 and p72 appear to have some overlapping functions, they clearly also have distinct, nonredundant functions. Furthermore, their ability to interact with a variety of different factors and act as multifunctional proteins has the potential to impact on several different processes, and alterations in expression or function of p68 and/or p72 could have profound implications for cancer development. However, their roles are likely to be context-dependent and both proteins have been reported to have pro-proliferation or even oncogenic functions as well as antiproliferative or tumor cosuppressor roles. Therefore, eludicating the precise role of these proteins in cancer is likely to be complex and to depend on the cellular environment and interacting factors. In this article, we review the many functions that have been attributed to p68 and p72 and discuss their potential roles in cancer development.

Heat shock factor-1 modulates p53 activity in the transcriptional response to DNA damage.

Here we define an important role for heat shock factor 1 (HSF1) in the cellular response to genotoxic agents. We demonstrate for the first time that HSF1 can complex with nuclear p53 and that both proteins are co-operatively recruited to p53-responsive genes such as p21. Analysis of natural and synthetic cis elements demonstrates that HSF1 can enhance p53-mediated transcription, whilst depletion of HSF1 reduces the expression of p53-responsive transcripts. We find that HSF1 is required for optimal p21 expression and p53-mediated cell-cycle arrest in response to genotoxins while loss of HSF1 attenuates apoptosis in response to these agents. To explain these novel properties of HSF1 we show that HSF1 can complex with DNA damage kinases ATR and Chk1 to effect p53 phosphorylation in response to DNA damage. Our data reveal HSF1 as a key transcriptional regulator in response to genotoxic compounds widely used in the clinical setting, and suggest that HSF1 will contribute to the efficacy of these agents.

DDX5 plays essential transcriptional and post-transcriptional roles in the maintenance and function of spermatogonia.

Mammalian spermatogenesis is sustained by mitotic germ cells with self-renewal potential known as undifferentiated spermatogonia. Maintenance of undifferentiated spermatogonia and spermatogenesis is dependent on tightly co-ordinated transcriptional and post-transcriptional mechanisms. The RNA helicase DDX5 is expressed by spermatogonia but roles in spermatogenesis are unexplored. Using an inducible knockout mouse model, we characterise an essential role for DDX5 in spermatogonial maintenance and show that Ddx5 is indispensable for male fertility. We demonstrate that DDX5 regulates appropriate splicing of key genes necessary for spermatogenesis. Moreover, DDX5 regulates expression of cell cycle genes in undifferentiated spermatogonia post-transcriptionally and is required for cell proliferation and survival. DDX5 can also act as a transcriptional co-activator and we demonstrate that DDX5 interacts with PLZF, a transcription factor required for germline maintenance, to co-regulate select target genes. Combined, our data reveal a critical multifunctional role for DDX5 in regulating gene expression programmes and activity of undifferentiated spermatogonia.

p68 (Ddx5) interacts with Runx2 and regulates osteoblast differentiation.

Runx2 is an essential transcription factor for osteoblast development from mesenchymal progenitors. Runx2 regulates gene expression by interacting with numerous transcription factors and co-activators to integrate signaling events within the nucleus. In this study we used affinity purification and proteomic techniques to identify novel Runx2 interacting proteins. One of these proteins is the DEAD box RNA helicase, p68 (Ddx5). p68 regulates many aspects of RNA expression, including transcription and splicing. p68 co-localized with Runx2 in punctate foci within the nucleus. In transcription assays, p68 functioned as a co-activator of Runx2, but its helicase activity was not essential for co-activation. In accordance, Runx2 transcriptional activity was muted in p68-suppressed cells. Surprisingly, osteoblast differentiation of the multipotent progenitor C2C12 cell line was accelerated by p68 suppression and Runx2 suppressed p68 expression in calvarial progenitor cells. Together these data demonstrate that p68 is a novel co-activator for Runx2, but it inhibits osteogenic differentiation of progenitor cells. Moreover Runx2 has an active role in regulating p68 levels in osteoblast precursors. Thus, crosstalk between Runx2 and p68 controls osteoblast specification and maturation at multiple levels.

DExD/H box RNA helicases: multifunctional proteins with important roles in transcriptional regulation.

The DExD/H box family of proteins includes a large number of proteins that play important roles in RNA metabolism. Members of this family have been shown to act as RNA helicases or unwindases, using the energy from ATP hydrolysis to unwind RNA structures or dissociate RNA-protein complexes in cellular processes that require modulation of RNA structures. However, it is clear that several members of this family are multifunctional and, in addition to acting as RNA helicases in processes such as pre-mRNA processing, play important roles in transcriptional regulation. In this review I shall concentrate on RNA helicase A (Dhx9), DP103 (Ddx20), p68 (Ddx5) and p72 (Ddx17), proteins for which there is a strong body of evidence showing that they play important roles in transcription, often as coactivators or corepressors through their interaction with key components of the transcriptional machinery, such as CREB-binding protein, p300, RNA polymerase II and histone deacetylases.

DDX17 nucleocytoplasmic shuttling promotes acquired gefitinib resistance in non-small cell lung cancer cells via activation of ß-catenin.

Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective for non-small cell lung cancer (NSCLC) patients with EGFR mutations, almost all these patients will eventually develop acquired resistance to EGFR-TKI. However, the molecular mechanisms responsible for gefitinib resistance remain still not fully understood. Here, we report that elevated DDX17 levels are observed in gefitinib-resistant NSCLC cells than gefitinib-sensitive cells. Upregulation of DDX17 enhances the gefitinib resistance, whereas DDX17-silenced cells partially restore gefitinib sensitivity. Mechanistically, we demonstrate that DDX17 disassociates the E-cadherin/ß-catenin complex, resulting in ß-catenin nuclear translocation and subsequently augmenting the transcription of ß-catenin target genes. Moreover, we identify two nuclear localization signal (NLS) and four nuclear export signal (NES) sequences mediated DDX17 nucleocytoplasmic shuttling via an exportin/importin-dependent pathways. Interruption of dynamic nucleocytoplasmic shuttling of DDX17 impairs DDX17-mediating the activation of ß-catenin and acquired resistance in NSCLC cells. In conclusion, our findings reveal a novel and important mechanism by which DDX17 contributes to acquired gefitinib resistance through exportin/importin-dependent cytoplasmic shuttling and followed by activation of ß-catenin, and DDX17 inhibition may be a promising strategy to overcome acquired resistance of gefitinib in NSCLC patients.

The loop structure and the RNA helicase p72/DDX17 influence the processing efficiency of the mice miR-132.

miRNAs are small RNAs that are key regulators of gene expression in eukaryotic organisms. The processing of miRNAs is regulated by structural characteristics of the RNA and is also tightly controlled by auxiliary protein factors. Among them, RNA binding proteins play crucial roles to facilitate or inhibit miRNA maturation and can be controlled in a cell, tissue and species-specific manners or in response to environmental stimuli. In this study we dissect the molecular mechanism that promotes the overexpression of miR-132 in mice over its related, co-transcribed and co-regulated miRNA, miR-212. We have shown that the loop structure of miR-132 is a key determinant for its efficient processing in cells. We have also identified a range of RNA binding proteins that recognize the loop of miR-132 and influence both miR-132 and miR-212 processing. The DEAD box helicase p72/DDX17 was identified as a factor that facilitates the specific processing of miR-132.

TP53 drives invasion through expression of its Δ133p53ß variant.

TP53 is conventionally thought to prevent cancer formation and progression to metastasis, while mutant TP53 has transforming activities. However, in the clinic, TP53 mutation status does not accurately predict cancer progression. Here we report, based on clinical analysis corroborated with experimental data, that the p53 isoform Δ133p53ß promotes cancer cell invasion, regardless of TP53 mutation status. Δ133p53ß increases risk of cancer recurrence and death in breast cancer patients. Furthermore Δ133p53ß is critical to define invasiveness in a panel of breast and colon cell lines, expressing WT or mutant TP53. Endogenous mutant Δ133p53ß depletion prevents invasiveness without affecting mutant full-length p53 protein expression. Mechanistically WT and mutant Δ133p53ß induces EMT. Our findings provide explanations to 2 long-lasting and important clinical conundrums: how WT TP53 can promote cancer cell invasion and reciprocally why mutant TP53 gene does not systematically induce cancer progression.

Phosphorylation of human estrogen receptor alpha at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera.

Estrogen receptor alpha (ERalpha) is a transcription factor that regulates expression of target genes in a ligand-dependent manner. Activation of gene expression is mediated by two transcription activation functions AF-1 and AF-2, which act in a promoter- and cell-specific manner. Whilst AF-2 activity is regulated by estrogen (E2) binding, the activity of AF-1 is additionally modulated by phosphorylation at several sites. One of these phosphorylation sites, serine 118 (S118) is of particular interest as its mutation significantly reduces ERalpha activity. Previous studies have shown that S118 can be phosphorylated by the ERK1/2 mitogen activated protein kinases (MAPK) and by the cyclin-dependent protein kinase Cdk7. In this study we use antisera that specifically recognize ERalpha phosphorylated at S118 to demonstrate that MAPK phosphorylates S118 in a ligand-independent manner, whereas Cdk7 mediates E2-induced phosphorylation of S118. E2 stimulation of S118 phosphorylation was observed within 10 min of its addition and was maximal at 10(-7) M E2. S118 phosphorylation was maximal at 30 min but then declined, such that by 180 min following E2 addition little S118 phosphorylation was evident. S118 phosphorylation was also induced by the partial estrogen antagonist 4-hydroxytamoxifen, but not by the complete antagonist ICI 182, 780. S118 phosphorylation upon addition of the MAPK inducers EGF or PMA followed the expected time courses. Finally, we show that ERalpha is phosphorylated at S118 in vivo using immunoblotting of extracts prepared from a series of ERalpha-positive breast tumours.