Human lung epithelial cells cultured in the presence of radon-emitting rock experience gene expression changes similar to those associated with tobacco smoke exposure.

Radon is the second leading cause of lung cancer, after tobacco smoke. While tobacco smoke-induced carcinogenesis has been studied extensively, far less is known about radon-induced carcinogenesis, particularly in relation to the influence of radon on gene expression. The objectives of the work described herein were to (a) determine if and how exposure to low dose radon-emitting rock influences cells, at the gene expression level, and (b) compare any gene expression changes resulting from the exposure to radon-emitting rock with those induced by exposure to tobacco smoke. Any potential radiation-induced gene expression changes were also compared to those induced by exposure to cannabis smoke, a non-carcinogen at low doses, used here as a smoke exposure comparator. Human lung epithelial cells were exposed to radon-emitting rock, tobacco smoke or cannabis smoke, over months, and RNA-sequencing was carried out. We found that the rock-exposed cells experienced significant gene expression changes, particularly of the gene AKR1C3, and that these changes, over time, increasingly reflected those associated with exposure to tobacco, but not cannabis, smoke. We postulate that the early gene expression changes common to both the radiation and tobacco smoke exposures constitute a related - potentially pre-carcinogenic - response. Our findings suggest that the length of time a dividing population of cells is exposed to a constant low concentration of radon (with a potential cumulative absorbed dose) could be an important risk parameter for neoplastic transformation/carcinogenesis.

Data relating to the transcriptomes of human lung epithelial cells exposed to radon-emitting rock, tobacco smoke or cannabis smoke.

Presented herein are RNA expression data linked to the exposure of human lung epithelial cells to either low dose radon-emitting rock, tobacco smoke or cannabis smoke. Two cell lines were used, one representing a 'normal' lung epithelial cell (BEAS-2B, derived from immortilized bronchial epithelial cells from a cadaver) and one representing a 'cancerous' lung epithelial cell (NCI-H1975, derived from a primary lung adenocarcinoma from a non-smoker). Control cells were cultured under standard conditions. Test cells were either (a) continuously cultured in the presence of pulverized uranium-containing rock emitting 38 Bq/m3 radon, or (b) exposed five days a week, to a 1:10,000 dilution of either tobacco or cannabis smoke from one cigarette. RNA was extracted from the cells at various time-points over a period of 1-17 weeks (7-140 days). cDNA libraries were prepared from the RNA, and the libraries were sequenced. Raw, aligned sequencing data, from 38 biosamples, are available through a public repository. Differential gene expression data, relating to control and test samples from various time-points, are linked to this article. Detailed analyses relating to these data can be found in the article "Human lung epithelial cells cultured in the presence of radon-emitting rock experience gene expression changes similar to those associated with tobacco smoke exposure" [1].

RBM10: Harmful or helpful-many factors to consider.

RBM10 is an RNA binding motif (RBM) protein expressed in most, if not all, human and animal cells. Interest in RBM10 is rapidly increasing and its clinical importance is highlighted by its identification as the causative agent of TARP syndrome, a developmental condition that significantly impacts affected children. RBM10's cellular functions are beginning to be explored, with initial studies demonstrating a tumor suppressor role. Very recently, however, contradictory results have emerged, suggesting a tumor promoter role for RBM10. In this review, we describe the current state of knowledge on RBM10, and address this dichotomy in RBM10 function. Furthermore, we discuss what may be regulating RBM10 function, particularly the importance of RBM10 alternative splicing, and the relationship between RBM10 and its paralogue, RBM5. As RBM10-related work is gaining momentum, it is critical that the various aspects of RBM10 molecular biology revealed by recent studies be considered moving forward. It is only if these recent advances in RBM10 structure and function are considered that a clearer insight into RBM10 function, and the disease states with which RBM10 mutation is associated, will be gained.

Splicing arrays reveal novel RBM10 targets, including SMN2 pre-mRNA.

BACKGROUND: RBM10 is an RNA binding protein involved in message stabilization and alternative splicing regulation. The objective of the research described herein was to identify novel targets of RBM10-regulated splicing. To accomplish this, we downregulated RBM10 in human cell lines, using small interfering RNAs, then monitored alternative splicing, using a reverse transcription-PCR screening platform. RESULTS: RBM10 knockdown (KD) provoked alterations in splicing events in 10-20% of the pre-mRNAs, most of which had not been previously identified as RBM10 targets. Hierarchical clustering of the genes affected by RBM10 KD revealed good conservation of alternative exon inclusion or exclusion across cell lines. Pathway annotation showed RAS signaling to be most affected by RBM10 KD. Of particular interest was the finding that splicing of SMN pre-mRNA, encoding the survival of motor neuron (SMN) protein, was influenced by RBM10 KD. Inhibition of RBM10 resulted in preferential expression of the full-length, exon 7 retaining, SMN transcript in four cancer cell lines and one normal skin fibroblast cell line. SMN protein is expressed from two genes, SMN1 and SMN2, but the SMN1 gene is homozygously disrupted in people with spinal muscular atrophy; as a consequence, all of the SMN that is expressed in people with this disease is from the SMN2 gene. Expression analyses using primary fibroblasts from control, carrier and spinal muscle atrophy donors demonstrated that RBM10 KD resulted in preferential expression of the full-length, exon 7 retaining, SMN2 transcript. At the protein level, upregulation of the full-length SMN2 was also observed. Re-expression of RBM10, in a stable RBM10 KD cancer cell line, correlated with a reversion of the KD effect, demonstrating specificity. CONCLUSION: Our work has not only expanded the number of pre-mRNA targets for RBM10, but identified RBM10 as a novel regulator of SMN2 alternative inclusion.

RBM10 promotes transformation-associated processes in small cell lung cancer and is directly regulated by RBM5.

Lung cancers are the leading cause of cancer-related deaths worldwide, with small cell lung cancer (SCLC) being the most aggressive type. At the time of diagnosis, SCLC has usually already metastasized, and an astonishing 95% of patients eventually succumb to the disease. This highlights the need for more effective SCLC screening and treatment options. Interestingly, the earliest and most frequent genetic alteration associated with lung cancers involves a lesion in the region to which the RNA binding protein RBM5 maps. We have recently shown that a decrease in RBM5 expression may be a key step in SCLC development, as RBM5 regulated many transformation-associated processes in SCLC cells. RBM5 is structurally and functionally similar to another RNA binding protein, RBM10. Both proteins have tumor-suppressor properties in a variety of cancer cell lines, and it has been suggested that RBM5 expression can influence RBM10. Due to their similarities, and the recent evidence that RBM10 is mutated in up to 21% of lung cancers, we hypothesized that RBM10 would share RBM5's tumor-suppressor properties in SCLC. Using transcriptome analysis and functional assays, we show, however, that RBM10's function was opposite to what we hypothesized; in the endogenously RBM5-null GLC20 SCLC cell line, RBM10 actually promoted cell proliferation and other transformation-associated processes. Using RNA immunoprecipitation followed by next generation sequencing (RIP-Seq) and Western blotting, we demonstrate that RBM5 post-transcriptionally regulated RBM10 expression via direct interaction with specific RBM10 splice variants. We propose a working model describing the impact of this interaction on cellular processes. Our results provide evidence that RBM10 expression, in RBM5-null tumors, may contribute to tumor growth and metastasis. Measurement of both RBM10 and RBM5 expression in clinical samples may therefore hold prognostic and/or potentially predictive value.

RBM5 reduces small cell lung cancer growth, increases cisplatin sensitivity and regulates key transformation-associated pathways.

Small cell lung cancer (SCLC) is the most aggressive type of lung cancer, with almost 95% of patients succumbing to the disease. Although RBM5, a tumor suppressor gene, is downregulated in the majority of lung cancers, its role in SCLC is unknown. Using the GLC20 SCLC cell line, which has a homozygous deletion encompassing the RBM5 gene locus, we established stable RBM5 expressing sublines and investigated the effects of RBM5 re-expression. Transcriptome and target identification studies determined that RBM5 directly regulates the cell cycle and apoptosis in SCLC cells, as well as significantly downregulates other important transformation-associated pathways such as angiogenesis and cell adhesion. RNA sequencing of paired non-tumor and tumor SCLC patient specimens showed decreased RBM5 expression in the tumors, and expression alterations in the majority of the same pathways that were altered in the GLC20 cells and sublines. Functional studies confirmed RBM5 expression slows SCLC cell line growth, and increases sensitivity to the chemotherapy drug cisplatin. Overall, our work demonstrates the importance of RBM5 expression to the non-transformed state of lung cells and the consequences of its deletion to SCLC development and progression.

Co- and post-transcriptional regulation of Rbm5 and Rbm10 in mouse cells as evidenced by tissue-specific, developmental and disease-associated variation of splice variant and protein expression levels.

BACKGROUND: Expression and function of the two RNA binding proteins and regulators of alternative splicing, RBM5 and RBM10, have largely been studied in human tissue and cell lines. The objective of the study described herein was to examine their expression in mouse tissue, in order to lay the framework for comprehensive functional studies using mouse models. METHODS: All RNA variants of Rbm5 and Rbm10 were examined in a range of normal primary mouse tissues. RNA and protein were examined in differentiating C2C12 myoblasts and in denervated and dystonin-deficient mouse skeletal muscle. RESULTS: All Rbm5 and Rbm10 variants examined were expressed in all mouse tissues and cell lines. In general, Rbm5 and Rbm10 RNA expression was higher in brain than in skin. RNA expression levels were more varied between cardiac and skeletal muscle, depending on the splice variant: for instance, Rbm10v1 RNA was higher in skeletal than cardiac muscle, whereas Rbm10v3 RNA was higher in cardiac than skeletal muscle. In mouse brain, cardiac and skeletal muscle, RNA encoding an approximately 17kDa potential paralogue of a small human RBM10 isoform was detected, and the protein observed in myoblasts and myotubes. Expression of Rbm5 and Rbm10 RNA remained constant during C2C12 myogenesis, but protein levels significantly decreased. In two muscle disease models, neither Rbm10 nor Rbm5 showed significant transcriptional changes, although significant specific alternative splicing changes of Rbm5 pre-mRNA were observed. Increased RBM10 protein levels were observed following denervation. CONCLUSIONS: The varied co-transcriptional and post-transcriptional regulation aspects of Rbm5 and Rbm10 expression associated with mouse tissues, myogenesis and muscle disease states suggest that a mouse model would be an interesting and useful model in which to study comprehensive functional aspects of RBM5 and RBM10.

Post-transcriptional regulation of Rbm5 expression in undifferentiated H9c2 myoblasts.

We previously examined the expression of Rbm5 during myoblast differentiation and found significantly more protein in the early stages of skeletal myoblast differentiation than during the later stages. We decided to determine if this elevated level was necessary for differentiation. Our hypothesis was that if high levels of Rbm5 protein expression were necessary for the initiation of skeletal myoblast differentiation, then inhibition of expression would prevent differentiation. Our long-term objective is to inhibit Rbm5 expression and examine the effect on H9c2 differentiation. Towards this end, stable knockdown clones and transient knockdown populations were generated. Expression analyses in H9c2 myoblasts demonstrated significant Rbm5 messenger RNA (mRNA) inhibition but, surprisingly, no effect on RBM5 protein levels. Expression of the Rbm5 paralogue Rbm10 was examined in order to (a) ensure no off-target knockdown effect, and (b) investigate any possible compensatory effects. RBM10 protein levels were found to be elevated, in both the clonal and transiently transfected populations. These results suggest that myoblast RBM5 expression is regulated by a process that includes RNA sequestration and/or controlled translation, and that (a) RBM5 function is compensated for by RBM10, and/or (b) RBM5 regulates RBM10 expression. We have developed a model to describe our findings, and suggest further experiments for testing its validity. Since upregulation of Rbm10 might compensate for downregulated Rbm5, and consequently might mask any potential knockdown effect, it could lead to incorrect conclusions regarding the importance of Rbm5 for differentiation. It is therefore imperative to determine how both RBM5 and RBM10 protein expression is regulated.

Insight into the role of alternative splicing within the RBM10v1 exon 10 tandem donor site.

BACKGROUND: RBM10 is an RNA binding protein involved in the regulation of transcription, alternative splicing and message stabilization. Mutations in RBM10, which maps to the X chromosome, are associated with TARP syndrome, lung and pancreatic cancers. Two predominant isoforms of RBM10 exist, RBM10v1 and RBM10v2. Both variants have alternate isoforms that differ by one valine residue, at amino acid 354 (RBM10v1) or 277 (RBM10v2). It was recently observed that a novel point mutation at amino acid 354 of RBM10v1, replacing valine with glutamic acid, correlated with preferential expression of an exon 11 inclusion variant of the proliferation regulatory protein NUMB, which is upregulated in lung cancer. FINDINGS: We demonstrate, using the GLC20 male-derived small cell lung cancer cell line - confirmed to have only one X chromosome - that the two (+/-) valine isoforms of RBM10v1 and RBM10v2 result from alternative splicing. Protein modeling of the RNA Recognition Motif (RRM) within which the alteration occurs, shows that the presence of valine inhibits the formation of one of the two α-helices associated with RRM tertiary structure, whereas the absence of valine supports the α-helical configuration. We then show 2-fold elevated expression of the transcripts encoding the minus valine RBM10v1 isoform in GLC20 cells, compared to those encoding the plus valine isoform. This expression correlates with preferential expression of the lung cancer-associated NUMB exon 11 inclusion variant. CONCLUSIONS: Our observations suggest that the ability of RBM10v1 to regulate alternative splicing depends, at least in part, on a structural alteration within the second RRM domain, which influences whether RBM10v1 functions to support or repress splicing. A model is presented.

Assessment of reference genes for real-time quantitative PCR gene expression normalization during C2C12 and H9c2 skeletal muscle differentiation.

Skeletal muscle differentiation occurs during muscle development and regeneration. To initiate and maintain the differentiated state, a multitude of gene expression changes occur. Accurate assessment of these differentiation-related gene expression changes requires good quality template, but more specifically, appropriate internal controls for normalization. Two cell line-based models used for in vitro analyses of muscle differentiation incorporate mouse C2C12 and rat H9c2 cells. In this study, we set out to identify the most appropriate controls for mRNA expression normalization during C2C12 and H9c2 differentiation. We assessed the expression profiles of Actb, Gapdh, Hprt, Rps12 and Tbp during C2C12 differentiation and of Gapdh and Rps12 during H9c2 differentiation. Using NormFinder, we validated the stability of the genes individually and of the geometric mean generated from different gene combinations. We verified our results using Myogenin. Our study demonstrates that using the geometric mean of a combination of specific reference genes for normalization provides a platform for more precise test gene expression assessment during myoblast differentiation than using the absolute expression value of an individual gene and reinforces the necessity of reference gene validation.

Differential downregulation of Rbm5 and Rbm10 during skeletal and cardiac differentiation.

RBM5 and RBM10 play an important role in transformed cells. This role includes influencing the alternative splicing and/or expression of factors involved in apoptosis and cell cycle arrest. To date, all apoptosis studies relating to RBM5 and RBM10 have been performed in transformed cell lines, potentially confounding mechanistic interpretation because of the many mutations present in this population. The objective of this study was to identify a physiologically relevant non-transformed system in which to examine the expression of RBM5 and RBM10 for future mechanistic and target identification studies. Our system of choice was H9c2 myoblast differentiation. Expression of Rbm5, Rbm10, and selected splice variants was examined by end-point or real-time PCR and Western blot. We determined that all of the examined Rbm5 and Rbm10 variants were expressed in H9c2 myoblasts and throughout skeletal and cardiac myoblast differentiation. Furthermore, expression was differentially downregulated in a lineage-specific manner, suggesting lineage-specific regulation and roles. There was no correlation between mRNA and protein expression for Rbm5, Rbm10v1, and Rbm10v2, suggesting post-transcriptional and/or post-translational regulation. The differentiation expression profiles suggest the products encoded by Rbm5 and Rbm10 play a more important role in skeletal than cardiac myoblast differentiation and influence similar processes in non-tumor, differentiating cells as in transformed cells. The data also suggest that full-length Rbm5 and Rbm10 play a less important role than their alternative splice variants and/or shorter protein isoforms. This work establishes myoblast differentiation as a relevant model in which to conduct functional studies regarding Rbm5 and Rbm10.

Downregulating activated epidermal growth factor receptor has no effect on RBM5 expression.

BACKGROUND: We were interested in determining how the tumor suppressor gene RBM5 is regulated in lung cancers. Previous studies suggested that the gene expression is related to histological subtype and smoking exposure, since in small cell lung cancers the RBM5 gene is deleted whereas in non-small cell lung carcinomas (NSCLC) RBM5 expression is reduced. Of particular interest was the recent finding that in lung adenocarcinomas, a histological subtype of NSCLC, smoking exposure correlated with mutational activity in the transforming growth factor alpha (TGF-a) signaling pathway. Lung adenocarcinomas from smokers were associated with activating KRAS mutations, whereas lung adenocarcinomas from never-smokers were associated with activating epidermal growth factor receptor (EGFR) mutations. We hypothesized that inhibition of RBM5 in lung adenocarcinomas is achieved indirectly via these activating mutations. The objective of the research described herein was to determine if EGFR activation and RBM5 expression are negatively correlated. METHODS: EGFR expression in the lung adenocarcinoma cell line NCI-H1975 was inhibited using small interfering RNA. RBM5 expression was examined by real-time quantitative polymerase chain reaction and Western blotting. RESULTS: Reduced EGFR expression did not correlate with any change in RBM5 expression at either the RNA or protein level. CONCLUSION: These results suggest that RBM5 expression is not directly regulated by EGFR in non-smoker related lung adenocarinomas, and that some other mechanism operates to inhibit either the expression or function of this potential tumour suppressor in lung cancers that retain the RBM5 gene.

The 3p21.3 tumor suppressor RBM5 resensitizes cisplatin-resistant human non-small cell lung cancer cells to cisplatin.

OBJECTIVE: Increasing RBM5 levels inhibit tumor cell growth and promote apoptosis. In this study, we investigated the role of RBM5 in the cisplatin resistance observed in human lung non-small cell lung cancer cells and evaluated the effect of RBM5 modulation on cell growth inhibition and apoptosis induced by cisplatin in the parental non-small cell lung cancer cells A549 and their cisplatin resistant counterparts, A549/DDP cells. METHODS: RBM5 mRNA and protein expression in the A549 and A549/DDP cells was analyzed by semi-quantitative RT-PCR and western blot. The A549/DDP cells were then transfected with a pcDNA3-RBM5 plasmid, and an RBM5-specific siRNA was transfected into A549 cells, prior to treatment with cisplatin. Semi-quantitative RT-PCR and western blot analyses were performed to confirm the expression of RBM5 mRNA or protein, and knockdown of RBM5 mRNA or protein, respectively. MTT assays were used to evaluate chemosensitivity to cisplatin. Apoptosis was assessed by DAPI nuclear staining and flow cytometric analysis with an Annexin-V-FITC apoptosis kit. Cytosolic cytochrome c, cleaved caspase-3 and cleaved caspase-9 were detected by western blot. RESULTS: The expression of RBM5 mRNA and protein was significantly reduced in the A549/DDP cells compared with the A549 cells. Exogenous expression of RBM5 by the pcDNA3-RBM5 resensitized the response of A549/DDP to cisplatin, resulting in a significant increase in tumor-suppressing activity induced by cisplatin. In contrast, downregulation of RBM5 with siRNA in the A549 cells inhibited cisplatin-induced apoptosis. We also found that the RBM5-enhanced chemosensitivity was associated with the release of cytochrome c into the cytosol, activation of caspase-9 and the downstream marker caspase-3. CONCLUSION: Our results demonstrate that RBM5 may serve as a biomarker with the ability to predict a response to cisplatin. It may also act as a prognostic indicator in lung cancer patients. Our findings suggest that there may be clinical utility for ectopic RBM5 such as enhancing and resensitizing nonresponders to cisplatin.

Differential expression of RBM5, EGFR and KRAS mRNA and protein in non-small cell lung cancer tissues.

BACKGROUND: RNA binding motif 5 (RBM5) is a tumor suppressor gene that modulates apoptosis through the regulation of alternative splicing of apoptosis-related genes. This study aimed to detect RBM5 expression in non-small cell lung cancer (NSCLC) and to associate RBM5 expression with clinicopathological data from NSCLC patients and EGFR and KRAS expression to better understand the potential role of RBM5 in NSCLC. METHOD: Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to detect expression of mRNA and protein, respectively, of RBM5, EGFR and KRAS in 120 paired non-tumor and tumor samples of NSCLC. RESULTS: The data showed that expression of RBM5 mRNA and protein was significantly reduced in NSCLC compared to normal tissues, whereas expression of both EGFR and KRAS genes was increased in NSCLC compared to normal tissues. Furthermore, the reduced RBM5 protein expression correlated with smoking status, tumor stage and lymph node metastasis of NSCLC, while overexpression of EGFR and KRAS proteins correlated with tumor stage and lymph node metastasis of NSCLC. Overexpression of KRAS protein was more frequent in smokers with NSCLC. In addition, expression of RBM5 mRNA and protein was negatively correlated with expression of EGFR and KRAS mRNA and protein in NSCLC tissues. CONCLUSION: This study suggests further evaluation of RBM5 expression is warranted for use of RBM5 as a biomarker for NSCLC patients.

RBM10 Modulates Apoptosis and Influences TNF-α Gene Expression.

Recent evidence suggests that protein encoded by the RNA Binding Motif 10 (RBM10) gene has the ability to modulate apoptosis. The objective of this study was to test this hypothesis by manipulating RBM10 expression levels and examining the downstream consequences. The results showed that transient overexpression of RBM10 correlated with significantly elevated levels of tumour necrosis factor alpha (TNF-α) mRNA and soluble TNF-α (sTNF-α) protein, and increased apoptosis (phosphatidyl serine exposure on the outer cell membrane and nuclear condensation). Stable RNA interference-mediated RBM10 knockdown clones were less susceptible to TNF-α-mediated apoptosis, and had decreased sTNF-α protein levels. Elevated levels of TNF-α associated with RBM10 overexpression resulted from increased TNF-α transcription, not TNF-α mRNA stabilization. These results suggest that RBM10 has the ability to modulate apoptosis, and that it does so via a mechanism involving alterations to TNFR super family-mediated signaling. These data provide the first direct evidence that human RBM10 can function as an apoptosis modulator and cytokine expression regulator.

p53 transactivation is involved in the antiproliferative activity of the putative tumor suppressor RBM5.

RBM5 (RNA-binding motif protein 5) is a nuclear RNA binding protein containing 2 RNA recognition motifs. The RBM5 gene is located at the tumor suppressor locus 3p21.3. Deletion of this locus is the most frequent genetic alteration in lung cancer, but is also found in other human cancers. RBM5 is known to induce apoptosis and cell cycle arrest but the molecular mechanisms of RBM5 function are poorly understood. Here, we show that RBM5 is important for the activity of the tumor suppressor protein p53. Overexpression of RBM5 enhanced p53-mediated inhibition of cell growth and colony formation. Expression of RBM5 augmented p53 transcriptional activity in reporter gene assays and resulted in increased mRNA and protein levels for endogenous p53 target genes. In contrast, shRNA-mediated knockdown of endogenous RBM5 led to decreased p53 transcriptional activity and reduced levels of mRNA and protein for endogenous p53 target genes. RBM5 affected protein, but not mRNA, levels of endogenous p53 after DNA damage suggest that RBM5 contributes to p53 activity through post-transcriptional mechanisms. Our results show that RBM5 contributes to p53 transcriptional activity after DNA damage and that growth suppression and apoptosis mediated by RBM5 are linked to activity of the tumor suppressor protein p53.

RBM5 as a putative tumor suppressor gene for lung cancer.

RBM5 is one member of a group of structurally related genes that includes RBM6 and RBM10. RBM10 maps to Xp11.23, and one allele is inactivated as a result of X chromosome inactivation. Both RBM5 and RBM6 map to 3p21.3, a tumor suppressor region that experiences loss of heterozygosity in the majority of lung cancers. Overexpression of RBM5, which encodes an RNA-binding protein involved in the regulation of alternative splicing and retards ascites associated tumor growth in immunocompromised mice, a phenomenon that may be related to an associated ability to modulate apoptosis. As part of our quest to gain a better understanding of how the proapoptotic activity of RBM5 might contribute to tumor suppressor function, we reviewed all the literature relating to RBM5 expression, with a focus on lung cancer. On the basis of the existing data, we suggest that-to more thoroughly assess the potential involvement of RBM5 as a lung cancer regulatory protein-more research is required regarding (a) the expression of not only full-length RBM5 but all of the alternate variants associated with the locus, in relation to histologic subtype and smoking history, and (b) the mutation status of various genes within the transforming growth factor-alpha signaling pathway, which may function to either directly or indirectly regulate RBM5 activity in RBM5-retaining lung cancers.

Identification and characterisation of a novel antisense non-coding RNA from the RBM5 gene locus.

Previous work from our lab identified a 326 base-pair (bp) cDNA, termed Je2, which mapped to the antisense strand of intron 6 of the putative tumour suppressor gene RBM5/LUCA-15/H37, and functioned as an apoptosis suppressor. The purpose of the work described herein was to determine if Je2 is part of a larger transcript, to clone that transcript and to examine its ability to modulate RBM5 expression. Northern blot analyses in conjunction with strand-specific reverse transcription and PCR revealed two novel transcripts, one antisense and one sense, that included Je2 as well as RBM5 intron 4 sequence. Using rapid amplification of cDNA ends (RACE), a novel 1.4 kb product including Je2 and intron 4 was cloned. In vitro transcription/translation did not result in the production of any protein product, from either strand. Genomic DNA analysis revealed the presence of a putative promoter region 5' to Je2, suggesting that the cloned 1.4 kb RACE product represents an antisense transcript that initiates within intron 6 and terminates within intron 4 of the RBM5 gene. This novel antisense, non-coding RNA was termed LUST, for LUCA-15-specific transcript. Ectopic overexpression of LUST coincided with elevated expression of the full-length RBM5+5+6 alternative RBM5 RNA splice variant, and reduced expression of the truncated, cytotoxic RBM5+5+6t/Clone 26 alternative RBM5 RNA splice variant. A model is proposed whereby LUST functions co-transcriptionally to mask a sense-strand regulatory sequence, common to both RBM5+5+6 and RBM5+5+6t/Clone 26 transcripts, that when unmasked results in premature termination of RBM5+5+6, thereby generating the cytotoxic truncated product, RBM5+5+6t/Clone 26. These results suggest that LUST is a novel, functional, non-coding RNA that plays a role in determining the apoptotic fate of a cell by regulating the expression of RBM5 splice variants.

Up-regulation of the proapoptotic caspase 2 splicing isoform by a candidate tumor suppressor, RBM5.

Similar to many genes involved in programmed cell death (PCD), the caspase 2 (casp-2) gene generates both proapoptotic and antiapoptotic isoforms by alternative splicing. Using a yeast RNA-protein interaction assay, we identified RBM5 (also known as LUCA-15) as a protein that binds to casp-2 pre-mRNA. In both transfected cells and in vitro splicing assay, RBM5 enhances the formation of proapoptotic Casp-2L. RBM5 binds to a U/C-rich sequence immediately upstream of the previously identified In100 splicing repressor element. Our mutagenesis experiments demonstrate that RBM5 binding to this intronic sequence regulates the ratio of proapoptotic/antiapoptotic casp-2 splicing isoforms, suggesting that casp-2 splicing regulation by RBM5 may contribute to its tumor suppressor activity. Our work has uncovered a player in casp-2 alternative splicing regulation and revealed a link between the alternative splicing regulator and the candidate tumor suppressor gene. Together with previous studies, our work suggests that splicing control of cell death genes may be an important aspect in tumorigenesis. Enhancing the expression or activities of splicing regulators that promote the production of proapoptotic splicing isoforms might provide a therapeutic approach to cancer.

RBM6-RBM5 transcription-induced chimeras are differentially expressed in tumours.

UNLABELLED: Transcription-induced chimerism, a mechanism involving the transcription and intergenic splicing of two consecutive genes, has recently been estimated to account for approximately 5% of the human transcriptome. Despite this prevalence, the regulation and function of these fused transcripts remains largely uncharacterised. RESULTS: We identified three novel transcription-induced chimeras resulting from the intergenic splicing of a single RNA transcript incorporating the two neighbouring 3p21.3 tumour suppressor locus genes, RBM6 and RBM5, which encode the RNA Binding Motif protein 6 and RNA Binding Motif protein 5, respectively. Each of the three novel chimeric transcripts lacked exons 3, 6, 20 and 21 of RBM6 and exon 1 of RBM5. Differences between the transcripts were associated with the presence or absence of exon 4, exon 5 and a 17 nucleotide (nt) sequence from intron 10 of RBM6. All three chimeric transcripts incorporated the canonical splice sites from both genes (excluding the 17 nt intron 10 insertion). Differential expression was observed in tumour tissue compared to non-tumour tissue, and amongst tumour types. In breast tumour tissue, chimeric expression was associated with elevated levels of RBM6 and RBM5 mRNA, and increased tumour size. No protein expression was detected by in vitro transcription/translation. CONCLUSION: These results suggest that RBM6 mRNA experiences altered co-transcriptional gene regulation in certain cancers. The results also suggest that RBM6-RBM5 transcription-induced chimerism might be a process that is linked to the tumour-associated increased transcriptional activity of the RBM6 gene. It appears that none of the transcription-induced chimeras generates a protein product; however, the novel alternative splicing, which affects putative functional domains within exons 3, 6 and 11 of RBM6, does suggest that the generation of these chimeric transcripts has functional relevance. Finally, the association of chimeric expression with breast tumour size suggests that RBM6-RBM5 chimeric expression may be a potential tumour differentiation marker.