LPS induces KH-type splicing regulatory protein-dependent processing of microRNA-155 precursors in macrophages.

The importance of post-transcriptional mechanisms for the regulation of the homoeostasis of the immune system and the response to challenge by microorganisms is becoming increasingly appreciated. We investigated the contribution of microRNAs (miRNAs) to macrophage activation induced by lipopolysaccharide (LPS). We first observed that Dicer knockout in bone marrow-derived macrophages (BMDMs) increases the LPS-induced expression of some inflammation mediators. miRNA microarray analysis in BMDMs revealed that LPS significantly induces the expression of a single miRNA, miR-155, and this induction depends on enhanced miR-155 maturation from its precursors. The single-strand RNA-binding protein KH-type splicing regulatory protein (KSRP) binds to the terminal loop of miR-155 precursors and promotes their maturation. Both inhibition of miR-155 and KSRP knockdown enhance the LPS-induced expression of select inflammation mediators, and the effect of KSRP knockdown is reverted by mature miR-155. Our studies unveil the existence of an LPS-dependent post-transcriptional regulation of miR-155 biogenesis. Once induced, miR-155 finely tunes the expression of select inflammation mediators in response to LPS.

Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling.

BACKGROUND: KSRP is a AU-rich element (ARE) binding protein that causes decay of select sets of transcripts in different cell types. We have recently described that phosphatidylinositol 3-kinase/AKT (PI3K-AKT) activation induces stabilization and accumulation of the labile beta-catenin mRNA through an impairment of KSRP function. RESULTS: Aim of this study was to identify additional KSRP targets whose stability and steady-state levels are enhanced by PI3K-AKT activation. First, through microarray analyses of the AU-rich transcriptome in pituitary alphaT3-1 cells, we identified 34 ARE-containing transcripts upregulated in cells expressing a constitutively active form of AKT1. In parallel, by an affinity chromatography-based technique followed by microarray analyses, 12 mRNAs target of KSRP, additional to beta-catenin, were identified. Among them, seven mRNAs were upregulated in cells expressing activated AKT1. Both steady-state levels and stability of these new KSRP targets were consistently increased by either KSRP knock-down or PI3K-AKT activation. CONCLUSION: Our study identified a set of transcripts that are targets of KSRP and whose expression is increased by PI3K-AKT activation. These mRNAs encode RNA binding proteins, signaling molecules and a replication-independent histone. The increased expression of these gene products upon PI3K-AKT activation could play a role in the cellular events initiated by this signaling pathway.

The RNA-binding protein KSRP promotes decay of beta-catenin mRNA and is inactivated by PI3K-AKT signaling.

Beta-catenin plays an essential role in several biological events including cell fate determination, cell proliferation, and transformation. Here we report that beta-catenin is encoded by a labile transcript whose half-life is prolonged by Wnt and phosphatidylinositol 3-kinase-AKT signaling. AKT phosphorylates the mRNA decay-promoting factor KSRP at a unique serine residue, induces its association with the multifunctional protein 14-3-3, and prevents KSRP interaction with the exoribonucleolytic complex exosome. This impairs KSRP's ability to promote rapid mRNA decay. Our results uncover an unanticipated level of control of beta-catenin expression pointing to KSRP as a required factor to ensure rapid degradation of beta-catenin in unstimulated cells. We propose KSRP phosphorylation as a link between phosphatidylinositol 3-kinase-AKT signaling and beta-catenin accumulation.

Genes regulated by hepatocyte growth factor as targets to sensitize ovarian cancer cells to cisplatin.

Advanced ovarian cancers are initially responsive to chemotherapy with platinum drugs but develop drug resistance in most cases. We showed recently that hepatocyte growth factor (HGF) enhances death of human ovarian cancer cell lines treated with cisplatin (CDDP) and that this effect is mediated by the p38 mitogen-activated protein kinase. In this work, we integrated genome-wide expression profiling, in silico data survey, and functional assays to identify transcripts regulated in SK-OV-3 ovarian cancer cells made more responsive to CDDP by HGF. Using oligonucleotide microarrays, we found that HGF pretreatment changes the transcriptional response to CDDP. Quantitative reverse transcription-PCR not only validated all the 15 most differentially expressed genes but also confirmed that they were primarily modulated by the combined treatment with HGF and CDDP and reversed by suppressing p38 mitogen-activated protein kinase activity. Among the differentially expressed genes, we focused functional analysis on two regulatory subunits of the protein phosphatase 2A, which were down-modulated by HGF plus CDDP. Decrease of each subunit by RNA interference made ovarian cancer cells more responsive to CDDP, mimicking the effect of HGF. In conclusion, we show that HGF and CDDP modulate transcription in ovarian cancer cells and that this transcriptional response is involved in apoptosis regulation. We also provide the proof-of-concept that the identified genes might be targeted to either increase the efficacy of chemotherapeutics or revert chemotherapy resistance.

Deletion in a (T)8 microsatellite abrogates expression regulation by 3'-UTR.

A high level of genetic instability might cause mutations to accumulate in tumours. Microsatellite instability (MSI), due to defects of the DNA mismatch repair system, affects in particular repeat sequences (microsatellites) scattered throughout the genome. By scanning transcriptome databases, we found that microsatellites in the human genome are less numerous in coding DNA than in the 3'-untranslated region (UTR), known to mediate control of gene expression. By mutation analysis, we identified a 1 bp deletion in a (T)(8) microsatellite embedded in the 1801 nucleotide long 3'-UTR of CEACAM1 gene, thought to be involved in tumour onset and progression. By Lentiviral Vector- mediated gene transfer, we showed that the wild-type but not the mutated CEACAM1 3'-UTR greatly decreased transgene expression at both mRNA and protein level. Messenger RNA abundance was fully regulated by the most 3' region of CEACAM1 3'-UTR. This region includes the (T)(8) microsatellite but not any known classified regulatory element. These data show that CEACAM1 3'-UTR contains non-canonical elements contributing to mRNA regulation, among which a short repeat sequence could play a critical regulatory function. This suggests that, in cancer cells, a single mutation in a 3'-UTR short microsatellite might strongly affect gene expression.

Amplification of repeat-containing transcribed sequences (ARTS): a transcriptome fingerprinting strategy to detect functionally relevant microsatellite mutations in cancer.

Cancer is a genetic disease caused by mutations in somatic cells. Those that carry advantageous mutations are favoured by natural selection. In most cancers, genetic instability increases mutation rate and facilitates cancer cell evolution. Microsatellite instability (MSI), due to defects of the DNA mismatch repair system, affects in particular repeat sequences (microsatellites) scattered throughout the genome. As mutations in expressed genes are more likely to be functional, we developed a procedure for the systematic identification of mutant repeat-containing expressed sequences (amplification of repeat-containing transcribed sequences, ARTS). The entire cell mRNA was converted into short double-stranded cDNA fragments linked to an adapter at both ends. Repeat-containing cDNA fragments were PCR amplified using the adapter-specific primer in combination with different arbitrary primers including the repeat. ARTS yielded discrete PCR products with lengths that were directly correlated to the lengths of the endogenous repeats. Comparison between ARTS products obtained from control cells and cancer cells with microsatellite instability (MSI+) revealed mRNAs carrying insertions or deletions at repeats. The subsequent sequencing allowed the identification of a series of frameshift-mutated mRNAs in MSI+ cancer cells, including the already described mutant BAX transcript. These data show that ARTS provides an unbiased genome-wide approach to the discovery of functionally relevant genes that could be affected by MSI in cancer.