The RNA helicase p68 (DDX5) is selectively required for the induction of p53-dependent p21 expression and cell-cycle arrest after DNA damage.

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 γ-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.

The DEAD-box protein p72 regulates ERalpha-/oestrogen-dependent transcription and cell growth, and is associated with improved survival in ERalpha-positive breast cancer.

The DEAD-box RNA helicases p68 (DDX5) and p72 (DDX17) have been shown to act as transcriptional co-activators for a diverse range of transcription factors, including oestrogen receptor-alpha (ERalpha). Here, we show that, although both proteins interact with and co-activate ERalpha in reporter gene assays, small interfering RNA-mediated knockdown of p72, but not p68, results in a significant inhibition of oestrogen-dependent transcription of endogenous ERalpha-responsive genes and oestrogen-dependent growth of MCF-7 and ZR75-1 breast cancer cells. Furthermore, immunohistochemical staining of ERalpha-positive primary breast cancers for p68 and p72 indicate that p72 expression is associated with an increased period of relapse-free and overall survival (P=0.006 and 0.016, respectively), as well as being inversely associated with Her2 expression (P=0.008). Conversely, p68 shows no association with relapse-free period, or overall survival, but it is associated with an increased expression of Her2 (P=0.001), AIB-1 (P<0.001) and higher tumour grade (P=0.044). Our data thus highlight a crucial role for p72 in ERalpha co-activation and oestrogen-dependent cell growth and provide evidence in support of distinct but important roles for both p68 and p72 in regulating ERalpha activity in breast cancer.

Modulation of transcriptional activity of the DEAD-box family of RNA helicases, p68 (Ddx5) and DP103 (Ddx20), by SUMO modification.

SUMO (small ubiquitin-related modifier) modification is known to have diverse effects on the activity of transcriptional regulators, often through alterations in their localization or interactions with other factors, and in most of the cases is associated with transcriptional repression. The DEAD-box family of RNA helicases includes a large number of proteins that are involved in various cellular processes. Several members are now known to be multifunctional and their activities are thought to be governed by interactions with other partners, which may be regulated by post-translational modifications. In the present paper, we shall briefly review recent evidence indicating that SUMO modification is important in modulating the activity of two DEAD-box proteins, p68 (Ddx5) and DP103 (Ddx20), which are known to be important transcriptional regulators.

SUMO modification of the DEAD box protein p68 modulates its transcriptional activity and promotes its interaction with HDAC1.

The nuclear protein p68 (also known as Ddx5) is a prototypic member of the 'DEAD box' family of RNA helicases, which has been shown to be abnormally expressed and modified in colorectal tumors and to function as an important transcriptional regulator. Here, we show that p68 is modified in vivo on a single site (K53) by the small ubiquitin-like modifier-2 (SUMO-2). We demonstrate that the SUMO E3 ligase PIAS1 interacts with p68 and enhances its SUMO modification in vivo. To determine the functional consequences of SUMO modification, we compared the transcriptional activity of p68 and a K53R mutant that could not be SUMO-modified. Our data show that SUMO modification enhances p68 transcriptional repression activity and inhibits the ability of p68 to function as a coactivator of p53. These findings may be explained by the ability of wild type, but not K53R p68, to alter the modification state of chromatin by recruitment of histone deacetylase 1 (HDAC1).

The highly related DEAD box RNA helicases p68 and p72 exist as heterodimers in cells.

The RNA helicases p68 and p72 are highly related members of the DEAD box family of proteins, sharing 90% identity across the conserved core, and have been shown to be involved in both transcription and mRNA processing. We previously showed that these proteins co-localise in the nucleus of interphase cells. In this study we show that p68 and p72 can interact with each other and self-associate in the yeast two-hybrid system. Co-immunoprecipitation experiments confirmed that p68 and p72 can interact in the cell and indicated that these proteins preferentially exist as hetero-dimers. In addition, we show that p68 can interact with NFAR-2, a protein that is also thought to function in mRNA processing. Moreover, gel filtration analysis suggests that p68 and p72 can exist in a variety of complexes in the cell (ranging from approximately 150 to approximately 400 kDa in size), with a subset of p68 molecules being in very large complexes (>2 MDa). The potential to exist in different complexes that may contain p68 and/or p72, together with a range of other factors, would provide the potential for these proteins to interact with different RNA substrates and would be consistent with recent reports implying a wide range of functions for p68/p72.

The nuclear DEAD box RNA helicase p68 interacts with the nucleolar protein fibrillarin and colocalizes specifically in nascent nucleoli during telophase.

The DEAD box protein, p68, is an established RNA-dependent ATPase and RNA helicase in vitro, but neither the physiological function of this protein nor the macromolecules with which it interacts are known. Using a yeast two-hybrid screen, we identified the nucleolar protein, fibrillarin, as a protein that interacts with p68. Coimmunoprecipitation experiments confirmed that p68 and fibrillarin can form complexes in cellular extracts, and deletion analysis identified regions in each protein responsible for mediating the interaction. Immunofluorescence studies using confocal microscopy revealed that, in interphase cells, while fibrillarin is predominantly nucleolar, p68 shows a diffuse granular nuclear staining but is largely excluded from the nucleoli. Strikingly, both proteins colocalize in nascent nucleoli during late telophase. These data are consistent with a role for p68 either in postmitotic nucleolar reassembly or in the activation of ribosomal DNA transcription/preribosomal RNA processing during telophase and suggest that differential subnuclear compartmentalization may be a mechanism by which interaction of p68 with fibrillarin is regulated in the cell.

Interaction of the Escherichia coli DEAD box protein DbpA with 23 S ribosomal RNA.

The Escherichia coli DEAD box protein DbpA is unique among the DEAD box family in that its ATPase activity is specifically stimulated by bacterial 23 S ribosomal RNA. We have analysed the interaction between DbpA and a specific region within 23 S rRNA (namely nucleotides 2508-2580) which stimulates full ATPase activity. Using electrophoretic mobility shift assays we show that DbpA binds to this "specific" region with greater efficiency than to other regions of 23 S rRNA, and is not competed off by a non-specific RNA or a mutant RNA in which one of the stem-loops has been disrupted. These data suggest that the secondary structure within this region of 23 S rRNA is important for its recognition and binding by DbpA. We have also examined the ability of DbpA to unwind RNA and show that the purified protein does not behave as an RNA helicase in vitro with the substrates tested.

p72: a human nuclear DEAD box protein highly related to p68.

P72, a novel human member of the DEAD box family of putative RNA-dependent ATPases and ATP-dependent RNA helicases was isolated from a HeLa cDNA library. The predicted amino acid sequence of p72 is highly homologous to that of the prototypic DEAD box protein p68. In addition to the conserved core domains characteristic of DEAD box proteins, p72 contains several N-terminal RGG RNA-binding domains and a serine/glycine rich C-terminus likely involved in mediating protein-protein interactions. A p72-specific probe detects two mRNAs of approximately 5300 and 9300 bases which, although ubiquitously expressed, show variability in their expression levels in different tissues. Purified recombinant p72 exhibits ATPase activity in the presence of a range of RNA moieties. Immunocytochemical studies of p68 and p72 show that these proteins localise to similar locations in the nucleus of HeLa cells, suggesting their involvement in a nuclear process.

The "DEAD box" protein DbpA interacts specifically with the peptidyltransferase center in 23S rRNA.

The Escherichia coli DEAD (Asp-Glu-Ala-Asp) box protein DbpA is a putative RNA helicase and established RNA-dependent ATPase and is the only member of the DEAD box protein family for which a specific RNA substrate, bacterial 23S rRNA, has been identified. We have investigated the nature of this specificity in depth and have localized by deletion mutagenesis and PCR a single region of 93 bases (bases 2496-2588) in 23S rRNA that is both necessary and sufficient for complete activation of ATPase activity of DbpA. This target region forms part of the peptidyltransferase center and includes many bases involved in interaction with the 3' terminal adenosines of both A- and P-site tRNAs. Deletion of stem loops within the 93-base segment abolished ATPase activation. Similarly, point mutations that disrupt base pairing within stem structures ablated stimulation of ATPase activity. These data are consistent with roles for DbpA either in establishing and/or maintaining the correct three-dimensional structure of the peptidyltransferase center in 23S rRNA during ribosome assembly or in the peptidyltransferase reaction.

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.

A comparative assessment of an automated blood microprocessor for fetal blood pH measurements in the labour ward.

A new fully automated, self-calibrating, blood pH analyser was compared with a standard reference instrument for the measurement of pH of fetal scalp and umbilical cord blood samples. The new instrument is reliable and simple to operate, the volume of blood needed for analysis is small and it does not require the availability of trained laboratory technicians for its use.

Major pressure of sores.

A survey of 999 patients within the Borders Health Board area showed that 9.4 per cent had at least one pressure sore. J.C. Barbenel, M.M. Jordan and S.M. Nicol of the bioengineering unit, University of Strathclyde, describe how they assessed the extent and distribution of the condition, estimated to cost the NHS pound 150m a year.

Incidence of pressure-sores in the Greater Glasgow Health Board area.

A survey was made by questionnaire of the prevalence and severity of pressure-sores on a given date among all hospital inpatients and all patients visited by a district nurse within the Greater Glasgow Health Board area. 8.8% of patients had a pressure-sore. Those aged 70 and over accounted for 70% of the patients with sores. Chairfast patients consistently had a higher pressure-sore frequency than bedfast patients of a similar degree of helplessness.