FRAP analysis of nucleocytoplasmic dynamics of the vitamin D receptor splice variant VDRB1: preferential targeting to nuclear speckles.
Ontology highlight
ABSTRACT:
SUBMITTER: Sunn KL
PROVIDER: S-EPMC1138958 | biostudies-other | 2005 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The oncogene amplified in breast cancer 1 (AIB1) is a nuclear receptor coactivator that plays a major role in the progression of various cancers. We previously identified a splice variant of AIB1 called AIB1-Δ4 that is overexpressed in breast cancer. Using mass spectrometry, we define the translation initiation of AIB1-Δ4 at Met(224) of the full-length AIB1 sequence and have raised an antibody to a peptide representing the acetylated N terminus. We show that AIB1-Δ4 is predominantly localized in the cytoplasm, although leptomycin B nuclear export inhibition demonstrates that AIB1-Δ4 can enter and traffic through the nucleus. Our data indicate an import mechanism enhanced by other coactivators such as p300/CBP. We report that the endogenously and exogenously expressed AIB1-Δ4 is recruited as efficiently as full-length AIB1 to estrogen-response elements of genes, and it enhances estrogen-dependent transcription more effectively than AIB1. Expression of an N-terminal AIB1 protein fragment, which is lost in the AIB1-Δ4 isoform, potentiates AIB1 as a coactivator. This suggests a model whereby the transcriptional activity of AIB1 is squelched by a repressive mechanism utilizing the N-terminal domain and that the increased coactivator function of AIB1-Δ4 is due to the loss of this inhibitory domain. Finally, we show, using Scorpion primer technology, that AIB1-Δ4 expression is correlated with metastatic capability of human cancer cell lines.
| S-EPMC3143642 | biostudies-literature
Project description:Background. Androgen receptor splice variant-7 (AR-V7) is a truncated form of the androgen receptor protein which lacks the ligand-binding domain, the target of enzalutamide and abiraterone, but remains constitutively active as a transcription factor. We hypothesized that detection of AR-V7 in circulating tumor cells (CTCs) from men with advanced prostate cancer would be associated with resistance to enzalutamide and abiraterone. Methods. We used quantitative reverse-transcription polymerase-chain-reaction (qRT-PCR) to interrogate CTCs for the presence or absence of AR-V7 from prospectively enrolled patients with metastatic castration-resistant prostate cancer initiating treatment with either enzalutamide or abiraterone. We examined associations between AR-V7 status and PSA response rates, PSA-progression-free-survival (PSA-PFS), clinical/radiographic-progression-free-survival (PFS), and overall survival (OS). Multivariable Cox regression analyses were performed to determine the independent effect of AR-V7 status on clinical outcomes. Results. Thirty-one enzalutamide-treated patients and thirty-one abiraterone-treated patients were enrolled, of which 38.7% and 19.4% had detectable AR-V7 from CTCs, respectively. Among men receiving enzalutamide, AR-V7–positive patients had inferior PSA response rates (0% vs 52.6%, P=0.004), PSA-PFS (median: 1.4 vs 6.0 months, P<0.001), PFS (median: 2.1 vs 6.1 months, P<0.001), and OS (median: 5.5 months vs not reached, P=0.002) compared to AR-V7–negative patients. Similarly, among men receiving abiraterone, AR-V7–positive patients had inferior PSA response rates (0% vs 68.0%, P=0.004), PSA-PFS (median: 1.3 months vs not reached, P<0.001), PFS (median: 2.3 months vs not reached, P<0.001), and OS (median: 10.6 months vs not reached, P=0.006). The negative prognostic impact of AR-V7 was maintained after adjusting for full-length-AR expression. Conclusions. Detection of AR-V7 in CTCs from patients with castration-resistant prostate cancer is associated with resistance to enzalutamide and abiraterone. A total of four metastatic castration-resistant prostate tumor samples from four patients were subjected to RNA-seq. Two samples were positive for androgen receptor splice variant 7 and the other two were negative for this variant.
2014-09-03 | E-GEOD-56701 | biostudies-arrayexpress
Project description:The androgen receptor (AR) is required for prostate development and is also a major driver of prostate cancer pathogenesis. Thus androgen deprivation therapy (ADT) is the mainstay of treatment for advanced prostate cancer. However, castration resistance due to expression of constitutively active AR splice variants is a significant challenge to prostate cancer therapy; little is known why effectiveness of ADT can only last for a relatively short time. In the present study, we show that PCGEM1 interacts with splicing factors heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and U2AF65, as determined by RNA precipitation and Western blot, suggesting a role for PCGEM1 in alternative splicing. In support of this possibility, PCGEM1 is correlated with AR3, a predominant and clinically important form of AR splice variants in prostate cancer. Moreover, androgen deprivation (AD) induces PCGEM1 and causes its accumulation in nuclear speckles. Finally, we show that the AD-induced PCGEM1 regulates the competition between hnRNP A1 and U2AF65 for AR pre-mRNA. AD promotes PCGEM1 to interact with both hnRNP A1 and U2AF65 with different consequences. While the interaction of PCGEM1 with hnRNP A1 suppresses AR3 by exon skipping, its interaction with U2AF65 promotes AR3 by exonization. Together, we demonstrate an AD-mediated AR3 expression involving PCGEM1 and splicing factors.
| S-EPMC4941255 | biostudies-literature
Project description:Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.
| S-EPMC3024783 | biostudies-literature
Project description:Background. Androgen receptor splice variant-7 (AR-V7) is a truncated form of the androgen receptor protein which lacks the ligand-binding domain, the target of enzalutamide and abiraterone, but remains constitutively active as a transcription factor. We hypothesized that detection of AR-V7 in circulating tumor cells (CTCs) from men with advanced prostate cancer would be associated with resistance to enzalutamide and abiraterone. Methods. We used quantitative reverse-transcription polymerase-chain-reaction (qRT-PCR) to interrogate CTCs for the presence or absence of AR-V7 from prospectively enrolled patients with metastatic castration-resistant prostate cancer initiating treatment with either enzalutamide or abiraterone. We examined associations between AR-V7 status and PSA response rates, PSA-progression-free-survival (PSA-PFS), clinical/radiographic-progression-free-survival (PFS), and overall survival (OS). Multivariable Cox regression analyses were performed to determine the independent effect of AR-V7 status on clinical outcomes. Results. Thirty-one enzalutamide-treated patients and thirty-one abiraterone-treated patients were enrolled, of which 38.7% and 19.4% had detectable AR-V7 from CTCs, respectively. Among men receiving enzalutamide, AR-V7–positive patients had inferior PSA response rates (0% vs 52.6%, P=0.004), PSA-PFS (median: 1.4 vs 6.0 months, P<0.001), PFS (median: 2.1 vs 6.1 months, P<0.001), and OS (median: 5.5 months vs not reached, P=0.002) compared to AR-V7–negative patients. Similarly, among men receiving abiraterone, AR-V7–positive patients had inferior PSA response rates (0% vs 68.0%, P=0.004), PSA-PFS (median: 1.3 months vs not reached, P<0.001), PFS (median: 2.3 months vs not reached, P<0.001), and OS (median: 10.6 months vs not reached, P=0.006). The negative prognostic impact of AR-V7 was maintained after adjusting for full-length-AR expression. Conclusions. Detection of AR-V7 in CTCs from patients with castration-resistant prostate cancer is associated with resistance to enzalutamide and abiraterone.
2014-09-03 | GSE56701 | GEO
Project description:The cGMP-stimulated PDE2A hydrolyzes both cyclic nucleotides, cGMP and cAMP. Three splice variants have been cloned from several species. Whereas PDE2A1 is soluble, PDE2A2 and PDE2A3 are membrane-bound enzymes of rat and bovine origin, respectively. To date it is unclear whether one species expresses all three variants. The splice variants only differ in their N termini, which likely determine the subcellular localization. However, the mechanism for membrane attachment remains unknown. Here, we show that myristoylation underlies membrane targeting of PDE2A3. The myristoylated enzyme was bound to plasma membranes, whereas mutation of the myristoyl recipient Gly2 prevented incorporation of [3H]myristate and turned PDE2A3 completely soluble. Additionally, Cys5 and to a minor extent Cys11 are required for targeting of PDE2A3. Substitution of the putatively palmitoylated cysteines partially solubilized the enzyme and led to an accumulation in the endoplasmic reticulum/Golgi compartment, as shown by fluorescence microscopy in HEK 293 and PC12 cells. In vivo, PDE2A is expressed in many tissues. By using newly generated antibodies selectively detecting the splice variants PDE2A3 or PDE2A1, respectively, we demonstrate on the protein level PDE2A3 expression in mouse brain where it is entirely membrane-associated and a widespread expression of soluble PDE2A1 in mouse tissues. We show that PDE2A localizes to synaptosomal membranes and in primary cultures of hippocampal neurons partially overlaps with the presynaptic marker synaptophysin as demonstrated by immunofluorescence. In sum, these results demonstrate dual acylation as mechanism targeting neuronal PDE2A3 to synapses thereby ensuring local control of cyclic nucleotides.
| S-EPMC2757980 | biostudies-literature
Project description:Inherited mutations in BRCA1 confer susceptibility to breast and ovarian neoplasms. However, the function of BRCA1 and the role of BRCA1 in noninherited cancer remain unknown. Characterization of alternately spliced forms of BRCA1 may identify functional regions; thus, we constructed expression vectors of BRCA1 and a splice variant lacking exon 11, designated BRCA1 delta 672-4095. Immunofluorescence studies indicate nuclear localization of BRCA1 but cytoplasmic localization of BRCA1 delta 672-4095. Two putative nuclear localization signals (designated NLS1 and NLS2) were identified in exon 11; immunofluorescence studies indicate that only NLS1 is required for nuclear localization. RNA analysis indicates the expression of multiple, tissue-specific forms of BRCA1 RNAs; protein analysis with multiple antibodies suggests that at least three BRCA1 isoforms are expressed, including those lacking exon 11. The results suggest that BRCA1 is a nuclear protein and raise the possibility that splicing is one form of regulation of BRCA1 function by alteration of the subcellular localization of expressed proteins.
| S-EPMC231769 | biostudies-other
Project description:Eukaryotic mRNAs undergo a cycle of transcription, nuclear export, and degradation. A major challenge is to obtain a global, quantitative view of these processes. Here we measured the genome-wide nucleocytoplasmic dynamics of mRNA in Drosophila cells by metabolic labeling in combination with cellular fractionation. By mathematical modeling of these data we determined rates of transcription, export and cytoplasmic decay for 5420 genes. We characterized these kinetic rates and investigated links with mRNA features, RNA-binding proteins (RBPs) and chromatin states. We found prominent correlations between mRNA decay rate and transcript size, while nuclear export rates are linked to the size of the 3'UTR. Transcription, export and decay rates are each associated with distinct spectra of RBPs. Specific classes of genes, such as those encoding cytoplasmic ribosomal proteins, exhibit characteristic combinations of rate constants, suggesting modular control. Binding of splicing factors is associated with faster rates of export, and our data suggest coordinated regulation of nuclear export of specific functional classes of genes. Finally, correlations between rate constants suggest global coordination between the three processes. Our approach provides insights into the genome-wide nucleocytoplasmic kinetics of mRNA and should be generally applicable to other cell systems.
| S-EPMC5557608 | biostudies-literature
Project description:PurposeThe thermodynamically favored complex between the nuclear vitamin D receptor (VDR) and 1α,25(OH)2-vitamin D3 (1,25D3) triggers a shift in equilibrium to favor VDR binding to DNA, heterodimerization with the nuclear retinoid x receptor (RXR) and subsequent regulation of gene transcription. The key amino acids and structural requirements governing VDR binding to nuclear coactivators (NCoA) are well defined. Yet very little is understood about the internal changes in amino acid flexibility underpinning the control of ligand affinity, helix 12 conformation and function. Herein, we use molecular dynamics (MD) to study how the backbone and side-chain flexibility of the VDR differs when a) complexed to 1α,25(OH)2-vitamin D3 (1,25D3, agonist) and (23S),25-dehydro-1α(OH)-vitamin D3-26,23-lactone (MK, antagonist); b) residues that form hydrogen bonds with the C25-OH (H305 and H397) of 1,25D3 are mutated to phenylalanine; c) helix 12 conformation is changed and ligand is removed; and d) x-ray water near the C1- and C3-OH groups of 1,25D3 are present or replaced with explicit solvent.MethodsWe performed molecular dynamic simulations on the apo- and holo-VDRs and used T-Analyst to monitor the changes in the backbone and side-chain flexibility of residues that form regions of the VDR ligand binding pocket (LBP), NCoA surface and control helix 12 conformation.ResultsThe VDR-1,25D3 and VDR-MK MD simulations demonstrate that 1,25D3 and MK induce highly similar changes in backbone and side-chain flexibility in residues that form the LBP. MK however did increase the backbone and side-chain flexibility of L404 and R274 respectively. MK also induced expansion of the VDR charge clamp (i.e. NCoA surface) and weakened the intramolecular interaction between H305---V418 (helix 12) and TYR401 (helix 11). In VDR_FF, MK induced a generally more rigid LBP and stronger interaction between F397 and F422 than 1,25D3, and reduced the flexibility of the R274 side-chain. Lastly the VDR MD simulations indicate that R274 can sample multiple conformations in the presence of ligand. When the R274 is extended, the β-OH group of 1,25D3 lies proximal to the backbone carbonyl oxygen of R274 and the side-chain forms H-bonds with hinge domain residues. This differs from the x-ray, kinked geometry, where the side-chain forms an H-bond with the 1α-OH group. Furthermore, 1,25D3, but not MK was observed to stabilize the x-ray geometry of R274 during the > 30 ns MD runs.ConclusionsThe MD methodology applied herein provides an in silico foundation to be expanded upon to better understand the intrinsic flexibility of the VDR and better understand key side-chain and backbone movements involved in the bimolecular interaction between the VDR and its' ligands.
| S-EPMC4215818 | biostudies-literature
Project description:Eukaryotic mRNAs undergo a cycle of transcription, nuclear export, and degradation. A major challenge is to obtain a global, quantitative view of these processes. Here we measured the genome-wide nucleocytoplasmic dynamics of mRNA in Drosophila cells by metabolic labeling in combination with cellular fractionation. By mathematical modeling of these data we determined rates of transcription, export and cytoplasmic decay for >5,000 genes. We characterized these kinetic rates and investigated links with mRNA features, RNA-binding proteins (RBPs) and chromatin states. We found prominent correlations between mRNA decay rate and transcript size, while nuclear export rates are linked to the size of the 3'UTR. Transcription, export and decay rates are each associated with distinct spectra of RBPs. Specific classes of genes, such as those encoding cytoplasmic ribosomal proteins, exhibit characteristic combinations of rate constants, suggesting modular control. Overall, transcription and decay rates have a major impact on transcript abundance, while nuclear export is of minor importance. Finally, correlations between rate constants suggest global coordination between the three processes. Our approach should be generally applicable to other cell systems and provides insights into the genome-wide nucleocytoplasmic kinetics of mRNA.
2017-07-31 | GSE83620 | GEO