Frances V. Fuller-Pace

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.

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

Estrogen receptor α (ERα) 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 ERα 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 ERα 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 ERα is phosphorylated at S118 in vivo using immunoblotting of extracts prepared from a series of ERα-positive breast tumours.

The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor

The DEAD box RNA helicase, p68, has been implicated in various cellular processes and has been shown to possess transcriptional coactivator function. Here, we show that p68 potently synergises with the p53 tumour suppressor protein to stimulate transcription from p53‐dependent promoters and that endogenous p68 and p53 co‐immunoprecipitate from nuclear extracts. Strikingly, RNAi suppression of p68 inhibits p53 target gene expression in response to DNA damage, as well as p53‐dependent apoptosis, but does not influence p53 stabilisation or expression of non‐p53‐responsive genes. We also show, by chromatin immunoprecipitation, that p68 is recruited to the p21 promoter in a p53‐dependent manner, consistent with a role in promoting transcriptional initiation. Interestingly, p68 knock‐down does not significantly affect NF‐κB activation, suggesting that the stimulation of p53 transcriptional activity is not due to a general transcription effect. This study represents the first report of the involvement of an RNA helicase in the p53 response, and highlights a novel mechanism by which p68 may act as a tumour cosuppressor in governing p53 transcriptional activity.

RNA helicases: modulators of RNA structure

RNA molecules play an essential role in many cellular processes, often as components of ribonucleoprotein complexes. Like proteins, RNA molecules adopt sequence-specific secondary and tertiary structures that are essential for function; alteration of these structures therefore provides a means of regulating RNA function. The discovery of DEAD box proteins, a large family of proteins that share several highly conserved motifs and have known or putative ATP-dependent RNA helicase activity, has provoked growing interest in the concept that regulation of RNA function may occur through local unwinding of complex RNA structures.

DbpA: a DEAD box protein specifically activated by 23s rRNA.

The Escherichia coli protein DbpA is a member of the ‘DEAD box’ family of putative RNA‐dependent ATPases and RNA helicases, so called because they share the highly conserved motif Asp‐Glu‐Ala‐Asp, together with several other conserved elements. We have investigated DbpA expression under conditions where an endogenous promoter is used. In this context, translation initiation does not occur at the previously identified AUG, but at an upstream, in‐frame GUG. Mutation of the GUG initiation codon to AUG virtually abolishes DbpA expression, suggesting an unusual translation initiation mechanism. Using an inducible overexpression plasmid, we have purified milligram quantities of DbpA to homogeneity and shown that the purified protein hydrolyses ATP in an RNA‐dependent manner. This ATPase activity is interesting in that, unlike that of other DEAD box proteins investigated to date, it absolutely requires a specific bacterial RNA, which we have identified as 23S rRNA. This observation is particularly significant since DbpA will bind other RNAs and DNA, but will only hydrolyse ATP in the presence of 23S rRNA.

The p68 and p72 DEAD box RNA helicases interact with HDAC1 and repress transcription in a promoter-specific manner

Backgroundp68 (Ddx5) and p72 (Ddx17) are highly related members of the DEAD box family and are established RNA helicases. They have been implicated in growth regulation and have been shown to be involved in both pre-mRNA and pre-rRNA processing. More recently, however, these proteins have been reported to act as transcriptional co-activators for estrogen-receptor alpha (ERα). Furthermore these proteins were shown to interact with co-activators p300/CBP and the RNA polymerase II holoenzyme. Taken together these reports suggest a role for p68 and p72 in transcriptional activation.ResultsIn this report we show that p68 and p72 can, in some contexts, act as transcriptional repressors. Targeting of p68 or p72 to constitutive promoters leads to repression of transcription; this repression is promoter-specific. Moreover both p68 and p72 associate with histone deacetylase 1 (HDAC1), a well-established transcriptional repression protein.ConclusionsIt is therefore clear that p68 and p72 are important transcriptional regulators, functioning as co-activators and/or co-repressors depending on the context of the promoter and the transcriptional complex in which they exist.

The RNA helicase p68 is a novel androgen receptor coactivator involved in splicing and is overexpressed in prostate cancer.

The androgen receptor (AR) is a member of the nuclear steroid hormone receptor family and is thought to play an important role in the development of both androgen-dependent and androgen-independent prostatic malignancy. Elucidating roles by which cofactors regulate AR transcriptional activity may provide therapeutic advancement for prostate cancer (PCa). The DEAD box RNA helicase p68 (Ddx5) was identified as a novel AR-interacting protein by yeast two-hybrid screening, and we sought to examine the involvement of p68 in AR signaling and PCa. The p68-AR interaction was verified by colocalization of overexpressed protein by immunofluorescence and confirmed in vivo by coimmunoprecipitation in the PCa LNCaP cell line. Chromatin immunoprecipitation in the same cell line showed AR and p68 recruitment to the promoter region of the androgen-responsive prostate-specific antigen (PSA) gene. Luciferase reporter, minigene splicing assays, and RNA interference (RNAi) were used to examine a functional role of p68 in AR-regulated gene expression, whereby p68 targeted RNAi reduced AR-regulated PSA expression, and p68 enhanced AR-regulated repression of CD44 splicing (P = 0.008). Tyrosine phosphorylation of p68 was found to enhance coactivation of ligand-dependent transcription of AR-regulated luciferase reporters independent of ATP-binding. Finally, we observe increased frequency and expression of p68 in PCa compared with benign tissue using a comprehensive prostate tissue microarray (P = 0.003; P = 0.008). These findings implicate p68 as a novel AR transcriptional coactivator that is significantly overexpressed in PCa with a possible role in progression to hormone-refractory disease.

Overexpression and poly-ubiquitylation of the DEAD-box RNA helicase p68 in colorectal tumours

The DEAD box RNA helicase, p68, is upregulated in exponentially growing cells and shows cell cycle-dependent changes in nuclear localization. Although some other DEAD box proteins have been implicated in cancer, there have been no reports of any link between p68 status and carcinogenesis. In the present study we have analysed specimens from 50 patients with colorectal adenocarcinomas, including cases in which an adenomatous polyp was also present, by immunohistochemistry and Western blotting. Our data indicate that p68 protein is consistently overexpressed in tumours as compared with matched normal tissue. Examination of the levels of p68 mRNA from both normal and tumour tissue showed no obvious specific increase in p68 mRNA levels in tumours nor any evidence of underlying mutations in the p68 coding region. Interestingly, however, the accumulated p68 appears to be poly-ubiquitylated, suggesting a possible defect in proteasome-mediated degradation in these tumours. This overexpression/ubiquitylation is observed in both pre-invasive and invasive lesions suggesting that the dysregulation of p68 expression occurs early during tumour development. Finally, we demonstrate that ubiquitylation of p68 occurs in cultured cells, thereby providing a model for the molecular analysis of this process and its potential role in tumorigenesis.

Expression of the 'dead box' RNA helicase p68 is developmentally and growth regulated and correlates with organ differentiation/maturation in the fetus

The human DEAD box protein p68 is an established RNA‐dependent ATPase and RNA helicase. p68 has been highly conserved in evolution and appears to be essential for normal growth, suggesting that this protein plays an important role in the cell. Although the biochemical activities of p68 are fairly well characterized, little is known about its biological function. This report shows that p68 is detectable in quiescent cell lines, but its expression is induced by serum, suggesting that this protein may play a role in cell growth. It is also shown that both p68 mRNA and protein are differentially expressed in adult tissues; in this case, however, the levels do not always correlate with proliferation status, suggesting that the regulation of expression in the animal may be different from that in cell lines. Finally, it is shown that p68 expression is developmentally regulated and appears to correlate with organ differentiation/maturation. These findings suggest that p68 expression may not simply reflect proliferation/differentiation status and that it appears to be regulated in a more complex way.

The DEAD box RNA helicases p68 (Ddx5) and p72 (Ddx17): novel transcriptional co-regulators

DEAD box [a motif named after its amino acid sequence (Asp-Glu-Ala-Asp)] RNA helicases are known to play key roles in all cellular processes that require modulation of RNA structure. However, in recent years, several of these proteins have been found to function in transcriptional regulation. In the present paper, we shall review the literature demonstrating the action of p68 and, where data are available, p72 as transcriptional co-regulators for a range of transcription factors, namely ERalpha (oestrogen receptor alpha), the tumour suppressor p53, the myogenic regulator MyoD and Runx2, a transcription factor essential for osteoblast development. We shall also discuss evidence indicating that, in some cases at least, p68 and p72 have distinct, non-redundant, roles.