CD46 splice variant enhances translation of specific mRNAs linked to an aggressive tumor cell phenotype in bladder cancer.
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ABSTRACT:
SUBMITTER: Zeng J
PROVIDER: S-EPMC7972933 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The RNA-binding protein TDP-43 is heavily implicated in neurodegenerative disease. Numerous patient mutations in TARDBP, the gene encoding TDP-43, combined with data from animal and cell-based models, imply that altered RNA regulation by TDP-43 causes Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. However, underlying mechanisms remain unresolved. Increased cytoplasmic TDP-43 levels in diseased neurons suggest a possible role in this cellular compartment. Here, we examined the impact on translation of overexpressing human TDP-43 and the TDP-43A315T patient mutant protein in motor neuron-like cells and primary cultures of cortical neurons. In motor-neuron like cells, TDP-43 associates with ribosomes without significantly affecting global translation. However, ribosome profiling and additional assays revealed enhanced translation and direct binding of Camta1, Mig12, and Dennd4a mRNAs. Overexpressing either wild-type TDP-43 or TDP-43A315T stimulated translation of Camta1 and Mig12 mRNAs via their 5'UTRs and increased CAMTA1 and MIG12 protein levels. In contrast, translational enhancement of Dennd4a mRNA required a specific 3'UTR region and was specifically observed with the TDP-43A315T patient mutant allele. Our data reveal that TDP-43 can function as an mRNA-specific translational enhancer. Moreover, since CAMTA1 and DENND4A are linked to neurodegeneration, they suggest that this function could contribute to disease.
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Project description:Platelets are anucleate cytoplasmic fragments that lack genomic DNA, but continue to synthesize protein using a pool of messenger RNAs (mRNAs), ribosomes, and regulatory small RNAs inherited from the precursor megakaryocyte (MK). The regulatory processes that shape the platelet transcriptome and the full scope of platelet translation have remained elusive. Using RNA sequencing (RNA-Seq) and ribosome profiling of primary human platelets, we show the platelet transcriptome encompasses a subset of transcripts detected by RNA-Seq analysis of in vitro-derived MK cells and that these platelet-enriched transcripts are broadly occupied by ribosomes. We use RNA-Seq of synchronized populations of in vitro-derived platelet-like particles to show that mRNA decay strongly shapes the nascent platelet transcriptome. Our data suggest that the decay of platelet mRNAs is slowed by the natural loss of the mRNA surveillance and ribosome rescue factor Pelota.
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Project description:Progression of conventional urothelial carcinoma of the bladder to a tumor with unique microscopic features referred to as micropapillary carcinoma is coupled with aggressive clinical behavior signified by a high propensity for metastasis to regional lymph nodes and distant organs resulting in shorter survival.To analyze the expression profile of micropapillary cancer and define its molecular features relevant to clinical behavior.We retrospectively identified 43 patients with micropapillary bladder cancers and a reference set of 89 patients with conventional urothelial carcinomas and performed whole-genome expression messenger RNA profiling.The tumors were segregated into distinct groups according to hierarchical clustering analyses. They were also classified according to luminal, p53-like, and basal categories using a previously described algorithm. We applied Ingenuity Pathway Analysis software (Qiagen, Redwood City, CA, USA) and gene set enrichment analysis for pathway analyses. Cox proportional hazards models and Kaplan-Meier methods were used to assess the relationship between survival and molecular subtypes. The expression profile of micropapillary cancer was validated for selected markers by immunohistochemistry on parallel tissue microarrays.We show that the striking features of micropapillary cancer are downregulation of miR-296 and activation of chromatin-remodeling complex RUVBL1. In contrast to conventional urothelial carcinomas that based on their expression can be equally divided into luminal and basal subtypes, micropapillary cancer is almost exclusively luminal, displaying enrichment of active peroxisome proliferator-activated receptor ? and suppression of p63 target genes. As with conventional luminal urothelial carcinomas, a subset of micropapillary cancers exhibit activation of wild-type p53 downstream genes and represent the most aggressive molecular subtype of the disease with the shortest survival. The involvement of miR-296 and RUVBL1 in the development of micropapillary bladder cancer was identified by the analyses of correlative associations of genome expression profiles and requires mechanistic validation.Micropapillary cancer evolves through the luminal pathway and is characterized by the activation of miR-296 and RUVBL1 target genes.Our observations have important implications for prognosis and for possible future development of more effective therapies for micropapillary bladder cancer.
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Project description:A key player in translation initiation is eIF4E, the mRNA 5' cap-binding protein. 4E-Transporter (4E-T) is a recently characterized eIF4E-binding protein, which regulates specific mRNAs in several developmental model systems. Here, we first investigated the role of its enrichment in P-bodies and eIF4E-binding in translational regulation in mammalian cells. Identification of the conserved C-terminal sequences that target 4E-T to P-bodies was enabled by comparison of vertebrate proteins with homologues in Drosophila (Cup and CG32016) and Caenorhabditis elegans by sequence and cellular distribution. In tether function assays, 4E-T represses bound mRNA translation, in a manner independent of these localization sequences, or of endogenous P-bodies. Quantitative polymerase chain reaction and northern blot analysis verified that bound mRNA remained intact and polyadenylated. Ectopic 4E-T reduces translation globally in a manner dependent on eIF4E binding its consensus Y30X4L site. In contrast, tethered 4E-T continued to repress translation when eIF4E-binding was prevented by mutagenesis of YX4L, and modestly enhanced the decay of bound mRNA, compared with wild-type 4E-T, mediated by increased binding of CNOT1/7 deadenylase subunits. As depleting 4E-T from HeLa cells increased steady-state translation, in part due to relief of microRNA-mediated silencing, this work demonstrates the conserved yet unconventional mechanism of 4E-T silencing of particular subsets of mRNAs.
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Project description:BackgroundEpigenetic silencing can extend to whole chromosomal regions in cancer. There have been few genome-wide studies exploring its involvement in tumorigenesis.MethodsWe searched for chromosomal regions affected by epigenetic silencing in cancer by using Affymetrix microarrays and real-time quantitative polymerase chain reaction to analyze RNA from 57 bladder tumors compared with normal urothelium. Epigenetic silencing was verified by gene re-expression following treatment of bladder cell lines with 5-aza-deoxycytidine, a DNA demethylating agent, and trichostatin A, a histone deacetylase inhibitor. DNA methylation was studied by bisulfite sequencing and histone methylation and acetylation by chromatin immunoprecipitation. Clustering was used to distinguish tumors with multiple regional epigenetic silencing (MRES) from those without and to analyze the association of this phenotype with histopathologic and molecular types of bladder cancer. The results were confirmed with a second panel of 40 tumor samples and extended in vitro with seven bladder cancer cell lines. All statistical tests were two-sided.ResultsWe identified seven chromosomal regions of contiguous genes that were silenced by an epigenetic mechanism. Epigenetic silencing was not associated with DNA methylation but was associated with histone H3K9 and H3K27 methylation and histone H3K9 hypoacetylation. All seven regions were concordantly silenced in a subgroup of 26 tumors, defining an MRES phenotype. MRES tumors exhibited a carcinoma in situ-associated gene expression signature (25 of 26 MRES tumors vs 0 of 31 non-MRES tumors, P < 10⁻¹⁴), rarely carried FGFR3 mutations (one of 26 vs 22 of 31 non-MRES tumors, P < 10⁻¹⁶), and contained 25 of 33 (76%) of the muscle-invasive tumors. Cell lines derived from aggressive bladder tumors presented epigenetic silencing of the same regions.ConclusionsWe have identified an MRES phenotype characterized by the concomitant epigenetic silencing of several chromosomal regions, which, in bladder cancer, is specifically associated with the carcinoma in situ gene expression signature.
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Project description:Deleterious mutations in the RUNX1 gene cause hereditary leukemia due to a rare syndrome called Familial platelet Disorder with Associated Myeloid Malignancy (FPDMM). We describe the characteristics of a family with FPDMM due to a novel RUNX1 mutation (L472X), located in the most 3-prime end of the gene reported to date. Our 36-year old proband presented with incidentally detected thrombocytopenia and a family history suggestive of FPDMM. Contrary to previously described families, affected members of our kindred express an eczematous phenotype, reportedly most severe in members who develop leukemia. Pedigree analysis shows that the L472X mutation tracks with thrombocytopenia, acute leukemia, and eczema. The L472X mutation produces a stably expressed RUNX1 protein product with a corresponding decrease in wild type RUNX1 expression. Our data supports the inclusion of eczema in the FPDMM phenotype and suggests the possibility that the RUNX1 L472X mutant causes the type of dominant negative affect that is associated with an elevated risk of leukemia in FPDMM families.
| S-EPMC3864030 | biostudies-literature
Project description:The present study aimed at identifying novel molecular cancer drug targets and biomarkers by analyzing the gene expression profiles of high-grade prostate cancer (PC), using a cDNA microarray combined with laser microbeam microdissection. A number of genes were identified that were transactivated in high-grade PC. First, a novel molecular target and diagnostic biomarker, shisa family member 2 (SHISA2), was identified as an overexpressed gene in high-grade PC cells. The reverse transcription-semi-quantitative polymerase chain reaction and immunohistochemical analysis validated the overexpression of SHISA2 (295 amino acids in length), specifically in high-grade PC cells with Gleason scores of between 8 and 10, relative to normal prostate epithelium. Knockdown of SHISA2 expression by short interfering RNA resulted in the marked suppression of PC cell viability. By contrast, exogenous SHISA2 expression in transfected cells promoted PC cell proliferation, indicating its oncogenic effects. Notably, as a result of cDNA microarray analysis, protein Wnt-5a (WNT5A) was focused upon and the expression of WNT5A was identified to be downregulated in SHISA2-knockdown. Western blot analysis validated significant downregulation of WNT5A by SHISA2-knockdown and upregulation of WNT5A by SHISA2 overexpression. The results of the present study indicated that SHISA2 may affect WNT5A synthesis. Furthermore, the secreted SHISA2 protein was determined in the culture medium of PC cells. We hypothesize that SHISA2 is involved in the regulation of WNT5A and in the aggressiveness of PC via the Wnt signaling pathway through WNT5A. Furthermore, SHISA2 may be a molecular target for cancer drugs, and a useful diagnostic biomarker for the prognosis and therapeutic effect in cancer.
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Project description:The expression of glutamine synthetase (GS), catalysing the ATP-dependent conversion of glutamate and ammonia into glutamine, is transcriptionally and post-transcriptionally regulated. The genomic structure of dog GS shown in the present study is basically similar to that of other mammals in that it is composed of seven exons and six introns. Using 5'-cRACE (where cRACE stands for circular rapid amplification of cDNA ends) and reverse transcriptase-PCR, we identified an additional exon (120 bp) in the first intron, designated in the present study as exon 1'. By means of alternative splicing, the GS gene produces an altered form of GS transcript with 5'-untranslated region (UTR) containing the exon 1'. This alternative transcript is abundantly expressed in brain, whereas it is found at lower levels in other tissues. In the human and mouse GS genes, extra exons are also found at the corresponding site of the intron 1 but with different sizes. An exon-trapping experiment for the GS gene in COS-7, Madin-Darby canine kidney and SK-N-SH cells revealed that the pattern of alternative splicing is variable in different cell types. The propensity of forming a secondary structure is predicted to be considerably higher in the presence of extra 5'-UTR, suggesting the possibility of a translational effect. To test this, we performed a reporter assay for fusions with different 5'-UTRs, demonstrating that the long form with extra 5'-UTR was translated 20- and 10-fold less than the short one in SK-N-SH and Neuro-2A cells respectively. Similarly, translations of human and mouse transcripts with extra 5'-UTRs were less efficient, showing 6-8-fold reductions in SK-N-SH cells. Furthermore, when we mutated an ATG sequence contained in the exon 1', the suppression of translation was partially relieved, suggesting that the negative regulation by an extra 5'-UTR is, to some extent, due to an abortive translation from the upstream ATG.
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Project description:Alternative splicing (AS) has been shown to participate in prostate cancer development and progression; however, a link between AS and prostate cancer health disparities has been largely unexplored. Here we report on the cloning of a novel splice variant of FGFR3 that is preferentially expressed in African American (AA) prostate cancer. This novel variant (FGFR3-S) omits exon 14, comprising 123 nucleotides that encode the activation loop in the intracellular split kinase domain. Ectopic overexpression of FGFR3-S in European American (EA) prostate cancer cell lines (PC-3 and LNCaP) led to enhanced receptor autophosphorylation and increased activation of the downstream signaling effectors AKT, STAT3, and ribosomal S6 compared with FGFR3-L (retains exon 14). The increased oncogenic signaling imparted by FGFR3-S was associated with a substantial gain in proliferative and antiapoptotic activities, as well as a modest but significant gain in cell motility. Moreover, the FGFR3-S-conferred proliferative and motility gains were highly resistant to the pan-FGFR small-molecule inhibitor dovitinib and the antiapoptotic gain was insensitive to the cytotoxic drug docetaxel, which stands in marked contrast with dovitinib- and docetaxel-sensitive FGFR3-L. In an in vivo xenograft model, mice injected with PC-3 cells overexpressing FGFR3-S exhibited significantly increased tumor growth and resistance to dovitinib treatment compared with cells overexpressing FGFR3-L. In agreement with our in vitro and in vivo findings, a high FGFR3-S/FGFR3-L expression ratio in prostate cancer specimens was associated with poor patient prognosis. IMPLICATIONS: This work identifies a novel FGFR3 splice variant and supports the hypothesis that differential AS participates in prostate cancer health disparities.
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Project description:Central nervous system myelination requires the synthesis of large amounts of myelin basic protein (MBP) at the axon-glia contact site. MBP messenger RNA (mRNA) is transported in RNA granules to oligodendroglial processes in a translationally silenced state. This process is regulated by the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binding to the cis-acting A2 response element (A2RE). Release of this repression of MBP mRNA translation is thus essential for myelination. Mice deficient in the Src family tyrosine kinase Fyn are hypomyelinated and contain reduced levels of MBP. Here, we identify hnRNP A2 as a target of activated Fyn in oligodendrocytes. We show that active Fyn phosphorylates hnRNP A2 and stimulates translation of an MBP A2RE-containing reporter construct. Neuronal adhesion molecule L1 binding to oligodendrocytes results in Fyn activation, which leads to an increase in hnRNP A2 phosphorylation. These results suggest that Fyn kinase activation results in the localized translation of MBP mRNA at sites of axon-glia contact and myelin deposition.
| S-EPMC2386098 | biostudies-literature