Alternative splicing variant of NRP/B promotes tumorigenesis of gastric cancer

Gastrointestinal cancer is associated with a high mortality rate. Here, we report that the splice variant of NRP/B contributes to tumorigenic activity in highly malignant gastric cancer through dissociation from the tumor repressor, HDAC5. NRP/B mRNA expression is significantly higher in the human gastric cancer tissues than in the normal tissues. Further, high levels of both the NRP/B splice variant and Lgr5, but not the full-length protein, are found in highly tumorigenic gastric tumor cells, but not in non-tumorigenic cells. The loss of NRP/B markedly inhibits cell migration and invasion, which reduces tumor formation in vivo. Importantly, the inhibition of alternative splicing increases the levels of NRP/B-1 mRNA and protein in AGS cells. The ectopic expression of full-length NRP/B exhibits tumor-suppressive activity, whereas NRP/B-2 induces the noninvasive human gastric cancer cells tumorigenesis. The splice variant NRP/B-2 which loses the capacity to interact with tumor repressors promoted oncogenic activity, suggesting that the BTB/POZ domain in the N-terminus has a crucial role in the suppression of gastric cancer. Therefore, the regulation of alternative splicing of the NRP/B gene is a potential novel target for the treatment of gastrointestinal cancer.     

Species-specific regulation of alternative splicing in the C-terminal region of the p53 tumor suppressor gene

Alternative splicing occurs in the C-terminal region of the p53 tumor suppressor gene between two alternative 3′ splice sites in intron 10. This alternative splicing event has been detected in murine cells, but not in rat or human tissues. In this paper, we have characterized the pattern of p53 alternative splicing in cell lines from five different species. Our results confirm that p53 alternative splicing is species-specific, being detected only in cell lines of rodent origin. Using transient transfection assays, we have established that the rat p53 gene undergoes efficient alternative splicing in both mouse and rat cell lines, thus demonstrating that it has all the necessary cis-acting sequences to be alternatively spliced. In contrast, we were unable to detect any usage of the human alternative 3′ splice site under the same experimental conditions. Thus, the low levels or absence of alternatively spliced p53 mRNA in rat and human cell lines seems to be the result of different mechanisms. Our results support the hypothesis that there are species-specific mechanisms implicated in the regulation of p53 activity.     

PIWIL4在人卵巢癌中的表达及其功能研究

采用半定量RT-PCR方法检测人卵巢透明细胞癌ES-2细胞中PIWIL1、PIWIL2、PIWIL3、PIWIL4 mRNA表达水平,以及PIWIL4在卵巢癌组织和癌旁正常组织中的表达情况．设计并化学合成针对PIWIL4的siRNA,用脂质体转染法将其转入ES-2细胞内,通过MTT和克隆形成实验,观察PIWIL4-siRNA对ES-2细胞生长活性和增殖能力的影响．半定量RT-PCR实验结果发现,ES-2细胞中,PIWIL4相对PIWIL1、PIWIL2、PIWIL3,其表达水平最高(P < 0.05),而且PIWIL4在卵巢癌组织中的表达明显高于癌旁正常组织(P < 0.01)．MTT实验和克隆形成实验结果显示,转染PIWIL4-siRNA的ES-2细胞生长活性和克隆形成率明显低于对照组(P < 0.05)．由此得出结论：PIWIL4在卵巢癌细胞系ES-2细胞表达较高,且它在卵巢癌组织中的表达明显上调,同时PIWIL4-siRNA可有效抑制ES-2细胞的生长活性和增殖能力,提示PIWIL4可能与卵巢癌发生、发展相关．     

Mammalian tissues defective in nonsense-mediated mRNA decay display highly aberrant splicing patterns

Background

Nonsense-mediated mRNA decay (NMD) affects the outcome of alternative splicing by degrading mRNA isoforms with premature termination codons. Splicing regulators constitute important NMD targets; however, the extent to which loss of NMD causes extensive deregulation of alternative splicing has not previously been assayed in a global, unbiased manner. Here, we combine mouse genetics and RNA-seq to provide the first in vivo analysis of the global impact of NMD on splicing patterns in two primary mouse tissues ablated for the NMD factor UPF2.

Results

We developed a bioinformatic pipeline that maps RNA-seq data to a combinatorial exon database, predicts NMD-susceptibility for mRNA isoforms and calculates the distribution of major splice isoform classes. We present a catalog of NMD-regulated alternative splicing events, showing that isoforms of 30% of all expressed genes are upregulated in NMD-deficient cells and that NMD targets all major splicing classes. Importantly, NMD-dependent effects are not restricted to premature termination codon+ isoforms but also involve an abundance of splicing events that do not generate premature termination codons. Supporting their functional importance, the latter events are associated with high intronic conservation.

Conclusions

Our data demonstrate that NMD regulates alternative splicing outcomes through an intricate web of splicing regulators and that its loss leads to the deregulation of a panoply of splicing events, providing novel insights into its role in core- and tissue-specific regulation of gene expression. Thus, our study extends the importance of NMD from an mRNA quality pathway to a regulator of several layers of gene expression.     

Alternative promoter usage of the membrane glycoprotein CD36

Background  

CD36 is a membrane glycoprotein involved in a variety of cellular processes such as lipid transport, immune regulation, hemostasis, adhesion, angiogenesis and atherosclerosis. It is expressed in many tissues and cell types, with a tissue specific expression pattern that is a result of a complex regulation for which the molecular mechanisms are not yet fully understood. There are several alternative mRNA isoforms described for the gene. We have investigated the expression patterns of five alternative first exons of the CD36 gene in several human tissues and cell types, to better understand the molecular details behind its regulation.     

The canine kallikrein-related peptidase 14: Structural characterization, alternative splicing and differential expression in mammary cancer

Human kallikrein-related peptidases (KLKs) represent a family of 15 serine proteases with diverse roles in many physiological and pathological processes, including carcinogenesis. In the dog, only two KLK genes are known; dKLK1 and canine arginine esterase. Recently, 12 other genes have been predicted using computational methods, but none of them has ever been experimentally validated in canine tissues. In this study we investigated the expression of Canis familiaris KLK14, (CANFA)KLK14, in normal and cancerous mammary tissues. First, it was demonstrated that the in-silico determined canine KLK14 mRNA (GenBank accession no: XM_541464) has been wrongfully predicted on its 5′-end (nucleotides 1–88). The (CANFA)KLK14 mRNA sequence presented here, has high homology to its human counterpart and exhibits all defining-KLK features. In addition to the classical form of the gene, five splice variants were also identified. The splicing events involved 5′-truncation or complete elimination of exon 4 and/or retention of intron I. All encoded protein products of the splice variants were predicted to be truncated and catalytically inactive. The classical form and variant 3 were almost ubiquitously expressed in both normal and neoplastic tissues. Variant 1 was predominantly detected in normal tissues. The classical form and variants 1 and 2 exhibited lower expression levels in tumor compared to normal tissues. Moreover, an Ile155Asn polymorphism was identified. This is the first report on the structural characterization, alternative splicing and tissue expression of canine KLK14 mRNA. These findings may form the basis for the establishment of comparative studies investigating KLK functions in health and disease using the dog as a model.     

Retinoic acid treatment and cell aggregation independently regulate alternative splicing in P19 cells during neural differentiation

To induce neural differentiation of P19 cells, two different treatments, RA (retinoic acid) and cell aggregation, are required. However, there has been no report that RA treatment alone or cell aggregation alone could control alternative splicing regulation in P19 cells. Therefore, we focused on alternative splicing effects by neural induction (RA treatment and/or cell aggregation) in P19 cells. We analysed the splicing patterns of several genes, including 5‐HT3R‐A (5‐hydroxytryptamine receptor), Actn1 (actinin alpha1), CUGBP2 (CUG‐binding protein) and PTB (polypyrimidine track‐binding protein), which showed different responses during the early neural induction of P19 cells. We show here that RA treatment alone changes the alternative splice mechanism of 5‐HT3R‐A. Cell aggregation alone controls alternative splicing regulation of Actn1. Both treatments (RA and cell aggregation) compensate and regulate the alternative splicing mechanism of CUGBP2. However, PTB is independent of RA and cell aggregation. Taken together, our results suggest that RA treatment and cell aggregation independently regulate the alternative splicing mechanism in the early stage of P19 cells during neural differentiation.     

Molecular Characterization of a cDNA Encoding Functional Human CLK4 Kinase and Localization to Chromosome 4q35

Phosphorylated serine- and arginine-rich (SR) proteins play an important role in the formation of spliceosomes, possibly controlling the regulation of alternative splicing. Enzymes that phosphorylate the SR proteins belong to the family of CDC2/CDC28-like kinases (CLK). Employing nucleotide sequence comparison of human expressed sequence tag sequences to the murine counterpart, we identified, cloned, and recombinantly expressed the human orthologue to the murine CLK4 cDNA. When fused to glutathione S-transferase, the catalytically active human CLK4 is able to autophosphorylate and to phosphorylate myelin basic protein, but not histone H2B as a substrate. Inspection of mRNA accumulation demonstrated gene expression in all human tissues, with the most prominent abundance in liver, kidney, brain, and heart. Using fluorescence in situ hybridization, the human CLK4 cDNA was localized to band q35 on chromosome 4.     

Ubiquitination Regulates Expression of the Serine/Arginine-rich Splicing Factor 1 (SRSF1) in Normal and Systemic Lupus Erythematosus (SLE) T Cells

T cells from patients with systemic lupus erythematosus (SLE) exhibit reduced expression of the critical T cell receptor (TCR)-associated CD3ζ signaling chain and are poor producers of the vital cytokine IL-2. By oligonucleotide pulldown and mass spectrometry discovery approaches, we identified the splicing regulator serine/arginine-rich splicing factor (SRSF) 1 or splicing factor 2/alternative splicing factor (SF2/ASF) to be important in the expression of CD3ζ chain. Importantly, increases in the expression of SRSF1 rescued IL-2 production in T cells from patients with SLE. In this study, we investigated the regulation of SRSF1 expression in resting and activated human T cells. We found that T cell stimulation induced a rapid and significant increase in mRNA expression of SRSF1; however, protein expression levels did not correlate with this increase. Co-engagement of CD28 induced a similar mRNA induction and reduction in protein levels. Proteasomal but not lysosomal degradation was involved in this down-regulation as evidenced by blocking with specific inhibitors MG132 and bafilomycin, respectively. Immunoprecipitation studies showed increased ubiquitination of SRSF1 in activated T cells. Interestingly, T cells from patients with SLE showed increased ubiquitination of SRSF1 when compared with those from healthy individuals. Our results demonstrate a novel mechanism of regulation of the splicing factor SRSF1 in human T cells and a potential molecular mechanism that controls its expression in SLE.     

Polyadenylation of Friend murine leukemia virus env‐mRNA is affected by its splicing

As splicing was previously found to be important for increasing Friend murine leukemia virus env‐mRNA stability and translation, we investigated whether splicing of env‐mRNA affected the poly(A) tail length using env expression vectors that yielded unspliced or spliced env‐mRNA. Incomplete polyadenylation was detected in a fraction of the unspliced env‐mRNA products in an env gene‐dependent manner, showing that splicing of Friend murine leukemia virus plays an important role in the efficiency of complete polyadenylation of env‐mRNA. These results suggested that the promotion of complete polyadenylation of env‐mRNA by splicing might partially explain up‐regulation of Env protein expression as a result of splicing.