Role of Auf1 in elimination of oxidatively damaged messenger RNA in human cells

In aerobically growing cells, in which reactive oxygen species are produced, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine, which induces alterations in gene expression. Here we show that the human Auf1 protein, also called HNRNPD, binds specifically to RNA containing this oxidized base and may be involved in cellular processes associated with managing the problems caused by RNA oxidation. Auf1-deficient cells were constructed from human HeLa and Nalm-6 lines using two different targeting procedures. Both types of Auf1-deficient cells are viable, but exhibit growth retardation. The stability of messenger RNA for four different housekeeping genes was determined in Auf1-deficient and -proficient cells, treated with or without hydrogen peroxide. The level of oxidized messenger RNA was considerably higher in Auf1-deficient cells than in Auf1-proficient cells. Auf1 may play a role in the elimination of oxidized RNA, which is required for the maintenance of proper gene expression under conditions of oxidative stress.     

Specific binding of 8-oxoguanine-containing RNA to polynucleotide phosphorylase protein

8-Oxoguanine, an oxidized form of guanine, has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors. To prevent such an outcome, organisms probably have a mechanism for recognizing RNA molecules carrying 8-oxoguanine and prevent them from entering into the cellular translational machinery. We now report that the Escherichia coli cell possesses proteins that bind specifically to RNA carrying 8-oxoguanine. On incubation with a cell-free extract, 8-oxoguanine-containing RNA is stable while normal RNA is degraded by cellular nucleases. The RNase protection assay and gel shift assay revealed that some proteins bind specifically to 8-oxoguanine-containing RNA, hence preventing nuclease attacks. Among the complexes that were detected, one with a 77 kDa protein exhibits tight binding between RNA and protein components. This protein was identified as polynucleotide phosphorylase, encoded by the pnp gene. pnp(-)() mutants are hyperresistant to paraquat, a drug that induces oxidative stress in the cell. Binding of Pnp protein to 8-oxoguanine-containing RNA would inhibit cell growth, probably due to withdrawal of such RNA from the translational machinery. The Pnp protein may, therefore, discriminate between an oxidized RNA molecule and a normal one, thus contributing a high fidelity of translation.     

LIN28A Modulates Splicing and Gene Expression Programs in Breast Cancer Cells

LIN28 is an evolutionarily conserved RNA-binding protein with critical functions in developmental timing and cancer. However, the molecular mechanisms underlying LIN28''s oncogenic properties are yet to be described. RNA-protein immunoprecipitation coupled with genome-wide sequencing (RIP-Seq) analysis revealed significant LIN28 binding within 843 mRNAs in breast cancer cells. Many of the LIN28-bound mRNAs are implicated in the regulation of RNA and cell metabolism. We identify heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a protein with multiple roles in mRNA metabolism, as a LIN28-interacting partner. Subsequently, we used a custom computational method to identify differentially spliced gene isoforms in LIN28 and hnRNP A1 small interfering RNA (siRNA)-treated cells. The results reveal that these proteins regulate alternative splicing and steady-state mRNA expression of genes implicated in aspects of breast cancer biology. Notably, cells lacking LIN28 undergo significant isoform switching of the ENAH gene, resulting in a decrease in the expression of the ENAH exon 11a isoform. The expression of ENAH isoform 11a has been shown to be elevated in breast cancers that express HER2. Intriguingly, analysis of publicly available array data from the Cancer Genome Atlas (TCGA) reveals that LIN28 expression in the HER2 subtype is significantly different from that in other breast cancer subtypes. Collectively, our data suggest that LIN28 may regulate splicing and gene expression programs that drive breast cancer subtype phenotypes.     

Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16

Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins to silencer elements in the exon and that down-regulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This article demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.     

Identification of PSF as a protein kinase Calpha-binding protein in the cell nucleus

Protein kinase C (PKC) isoforms are present in the cell nucleus in diverse cell lines and tissues. Since little is known about proteins interacting with PKC inside the cell nucleus, we used Neuro-2a neuroblastoma cells, in which PKCalpha is present in the nucleus, to screen for nuclear binding partners for PKC. Applying overlay assays, we detected several nuclear proteins which bind to PKCalpha. Specificity of binding was shown by its dependence on PKC activation by phorbol ester, calcium, and phosphatidylserine. The PKC-binding proteins were partially purified and analyzed by microsequencing and mass spectrometry. Four proteins could be identified: PTB-associated splicing factor (PSF), p68 RNA helicase, and the heterogeneous nuclear ribonucleoprotein (hnRNP) proteins A3 and L. In the case of PSF, binding to PKC could also be demonstrated in a GST-pull-down assay using GST-PKCalpha, expressed in insect cells. Phosphorylation experiments revealed that PSF is a weak in vitro substrate for PKCalpha.     

HNRNPC regulates RhoA to induce DNA damage repair and cancer‐associated fibroblast activation causing radiation resistance in pancreatic cancer

Pancreatic cancer (PC) is one of the most lethal types of cancer due to its asymptomatic nature in the early stages and consequent late diagnosis. Its mortality rate remains high despite advances in treatment strategies, which include a combination of surgical resection and adjuvant therapy. Although these approaches may have a positive effect on prognosis, the development of chemo‐ and radioresistance still poses a significant challenge for successful PC treatment. Heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) and RhoA have been implicated in the regulation of tumour cell proliferation and chemo‐ and radioresistance. Our study aims to investigate the mechanism for HNRNPC regulation of PC radiation resistance via the RhoA pathway. We found that HNRNPC and RhoA mRNA and protein expression levels were significantly higher in PC tissues compared to adjacent non‐tumour tissue. Furthermore, high HNRNPC expression was associated with poor patient prognosis. Using HNRNPC overexpression and siRNA interference, we demonstrated that HNRNPC overexpression promoted radiation resistance in PC cells, while HNRNPC knockdown increased radiosensitivity. However, silencing of RhoA expression was shown to attenuate radiation resistance caused by HNRNPC overexpression. Next, we identified RhoA as a downstream target of HNRNPC and showed that inhibition of the RhoA/ROCK2‐YAP/TAZ pathway led to a reduction in DNA damage repair and radiation resistance. Finally, using both in vitro assays and an in vivo subcutaneous tumour xenograft model, we demonstrated that RhoA inhibition can hinder the activity of cancer‐related fibroblasts and weaken PC radiation resistance. Our study describes a role for HNRNPC and the RhoA/ROCK2‐YAP/TAZ signalling pathways in mediating radiation resistance and provides a potential therapeutic target for improving the treatment of PC.     

Differential liquid phase proteomic analysis of the effect of selenium supplementation in LNCaP cells

The effect of 100 nM sodium selenite supplementation was studied on LNCaP cells by a proteomic approach, on ProteomeLab PF 2D platform. Proteins were separated by liquid phase bi-dimensional chromatography and analyzed by pair-wise alignment of peaks to detect those differentially expressed. Differential expression threshold was set at a twice difference level and proteins matching this criterion were identified by MALDI-TOF and confirmed by ESI-ion trap MS/MS. Not all differentially expressed proteins found by PF 2D could be identified by MS analysis, the sensitivity of which emerging as the limiting factor. Thus, only the most abundant proteins, differently expressed following selenium supplementation, were identified. We positively showed an increase of expression of thioredoxin reductase 1, enolase 1, phosphoglycerate mutase 1, glyceraldehyde-3-phosphate dehydrogenase, heterogeneous nuclear ribonucleoprotein A2/B1, isoform A2, Ras-GTPase-activating protein SH3-domain-binding protein and Keratin 18 and a decrease of expression of peroxiredoxin 1 and heat shock protein 70, protein 8, isoform 1. Results are consistent, at least in part, with the less oxidant environment brought about by the synthesis of Se-dependent peroxidases, keeping low the steady-state concentration of hydrogen peroxide.     

Binding capacity of human YB-1 protein for RNA containing 8-oxoguanine

8-oxoguanine (8-oxo-7,8-dihydroguanine) is generated in the cellular nucleotide pool as well as in nucleic acids, by the action of oxygen radicals produced in cells. 8-oxoguanine has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors. To prevent such an outcome, organisms should have mechanisms for preventing the misincorporation of 8-oxoguanine-containing nucleotide into RNA and for removing 8-oxoguanine-containing RNA from processes of translation. We now report that mammalian Y box-binding protein 1 (YB-1 protein) possesses the activity to bind specifically to RNA containing 8-oxoguanine. On incubation with a purified preparation of YB-1 protein, 8-oxoguanine-containing RNA forms stable complexes with the protein while normal RNA scarcely forms such a complex. Using a series of deletion mutants which produce altered forms of YB-1 protein lacking some parts of the sequence, domains of the protein necessary for RNA binding were identified. Escherichia coli cells expressing normal or truncated forms of YB-1 protein with the binding capacity acquire resistance against paraquat, a drug that induces oxidative stress in cells, whereas cells with truncated proteins lacking such an activity do not. YB-1 protein may disturb the bacterial system in recognizing oxidatively damaged RNA, thus exerting a dominant negative effect on cell growth. We propose that YB-1 protein may discriminate the oxidized RNA molecule from normal ones, thus contributing to the high fidelity of translation in cells.     

Human polynucleotide phosphorylase protein in response to oxidative stress

8-Oxo-7,8-dihydroguanine (8-oxoGua) is generated in nucleic acids as well as in their precursors due to the actions of oxygen radicals produced through a normal cellular metabolism. Since oxidized guanine can pair with both cytosine and adenine, it causes alterations in the phenotypic expression when it is present in RNA. To prevent such an outcome, organisms must have some mechanism for eliminating such oxidized guanine nucleotides from RNA and its precursors. In mammalian cells, MTH1 and NUDT5 proteins degrade 8-oxoGTP and 8-oxoGDP to 8-oxoGMP, which is an unusable form for RNA synthesis. In a search for proteins functioning at the RNA level, polynucleotide phosphorylase (PNP) protein has been suggested to be a good candidate for such a role. The human PNP protein has an ability to bind specifically to RNA containing 8-oxoGua. When human cells are exposed to agents that induce oxidative stress, such as hydrogen peroxide and menadion, the amounts of PNP protein decrease rapidly while amounts of other proteins in the cells do not change after such treatments. No specific decrease in the PNP protein level is observed when cells are treated with ACNU and cycloheximide at doses sufficient to provide the same degree of growth suppression. These results imply that the PNP protein might thus play a role in excluding oxidized forms of RNA from the translation mechanism.     

A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response

Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.     

Differential Subcellular Distributions and Trafficking Functions of hnRNP A2/B1 Spliceoforms

Trafficking of mRNA molecules from the nucleus to distal processes in neural cells is mediated by heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 trans‐acting factors. Although hnRNP A2/B1 is alternatively spliced to generate four isoforms, most functional studies have not distinguished between these isoforms. Here, we show, using isoform‐specific antibodies and isoform‐specific green fluorescent protein (GFP)‐fusion expression constructs, that A2b is the predominant cytoplasmic isoform in neural cells, suggesting that it may play a key role in mRNA trafficking. The differential subcellular distribution patterns of the individual isoforms are determined by the presence or absence of alternative exons that also affect their dynamic behavior in different cellular compartments, as measured by fluorescence correlation spectroscopy. Expression of A2b is also differentially regulated with age, species and cellular development. Furthermore, coinjection of isoform‐specific antibodies and labeled RNA into live oligodendrocytes shows that the assembly of RNA granules is impaired by blockade of A2b function. These findings suggest that neural cells modulate mRNA trafficking by regulating alternative splicing of hnRNP A2/B1 and controlling expression levels of A2b, which may be the predominant mediator of cytoplasmic‐trafficking functions. These findings highlight the importance of considering isoform‐specific functions for alternatively spliced proteins.