scholarly journals Exonic Splicing Enhancer-Dependent Selection of the Bovine Papillomavirus Type 1 Nucleotide 3225 3′ Splice Site Can Be Rescued in a Cell Lacking Splicing Factor ASF/SF2 through Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway

2003 ◽  
Vol 77 (3) ◽  
pp. 2105-2115 ◽  
Author(s):  
Xuefeng Liu ◽  
Akila Mayeda ◽  
Mingfang Tao ◽  
Zhi-Ming Zheng
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Viral Rna ◽  
Splicing Factor ◽  
Rna Expression ◽  
Bovine Papillomavirus ◽  
Phosphatidylinositol 3 Kinase ◽  
Selection Of ◽  
Phosphatidylinositol 3

ABSTRACT Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5′ splice site alternatively splices to a proximal 3′ splice site (at nucleotide 3225) to express L2 or to a distal 3′ splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF/SF2 (alternative splicing factor/splicing factor 2) binding sites, are located between the two 3′ splice sites and have been identified as regulating alternative 3′ splice site usage. The present report demonstrates for the first time that ASF/SF2 is required under physiological conditions for the expression of BPV-1 late RNAs and for selection of the proximal 3′ splice site for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline-repressible promoter. Depletion of ASF/SF2 from the cells by tetracycline greatly decreased viral RNA expression and RNA splicing at the proximal 3′ splice site while increasing use of the distal 3′ splice site in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-immunoglobulin M-B-cell receptor cross-linking rescued viral RNA expression and splicing at the proximal 3′ splice site and enhanced Akt phosphorylation and expression of the phosphorylated serine/arginine-rich (SR) proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific phosphatidylinositol 3-kinase/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3′ splice site, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the phosphatidylinositol 3-kinase/Akt pathway and is associated with the enhanced expression of other SR proteins.

scholarly journals Decreased L-Type Ca2+ Current in Cardiac Myocytes of Type 1 Diabetic Akita Mice Due to Reduced Phosphatidylinositol 3-Kinase Signaling

Diabetes ◽  
2007 ◽  
Vol 56 (11) ◽  
pp. 2780-2789 ◽  
Author(s):  
Z. Lu ◽  
Y.-P. Jiang ◽  
X.-H. Xu ◽  
L. M. Ballou ◽  
I. S. Cohen ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Kinase Signaling ◽  
Phosphatidylinositol 3 Kinase ◽  
Akita Mice ◽  
Phosphatidylinositol 3

Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo

1988 ◽  
Vol 8 (4) ◽  
pp. 1558-1569
Author(s):  
P E Cizdziel ◽  
M de Mars ◽  
E C Murphy
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Mrna Splicing ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Exon Ligation ◽  
Branch Point Sequence ◽  
Infected Cells ◽  
Low Efficiency

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.

scholarly journals RNA Splicing at Human Immunodeficiency Virus Type 1 3′ Splice Site A2 Is Regulated by Binding of hnRNP A/B Proteins to an Exonic Splicing Silencer Element

2001 ◽  
Vol 75 (18) ◽  
pp. 8487-8497 ◽  
Author(s):  
Patricia S. Bilodeau ◽  
Jeffrey K. Domsic ◽  
Akila Mayeda ◽  
Adrian R. Krainer ◽  
C. Martin Stoltzfus
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Splice Site ◽  
Rna Splicing ◽  
Consensus Sequence ◽  
Mrna Levels ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3′ splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1B, A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore splicing repression. The results suggest that coordinate repression of HIV-1 RNA splicing is mediated by members of the hnRNP A/B protein family.

scholarly journals Aquaporins and Fetal Membranes From Diabetic Parturient Women: Expression Abnormalities and Regulation by Insulin

2015 ◽  
Vol 100 (10) ◽  
pp. E1270-E1279 ◽  
Author(s):  
Damien Bouvier ◽  
Marion Rouzaire ◽  
Geoffroy Marceau ◽  
Cécile Prat ◽  
Bruno Pereira ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Kinase Inhibitor ◽  
Fetal Membranes ◽  
Phosphatidylinositol 3 Kinase ◽  
Intracellular Signaling Pathway ◽  
The Impact ◽  
Phosphatidylinositol 3

Context: During pregnancy, aquaporins (AQPs) expressed in fetal membranes are essential for controlling the homeostasis of the amniotic volume, but their regulation by insulin was never explored in diabetic women. Objective: The aim of our study was to investigate the involvement of AQPs 1, 3, 8, and 9 expressed in fetal membranes in diabetic parturient women and the control of their expression by insulin. Design and Participants: From 129 fetal membranes in four populations (controls, type 1, type 2 [T2D], and gestational diabetes [GD]), we established an expression AQP profile. In a second step, the amnion was used to study the control of the expression and functions of AQPs 3 and 9 by insulin. Main Outcomes and Measures: The expression of transcripts and proteins of AQPs was studied by quantitative RT-PCR and ELISA. We analyzed the regulation by insulin of the expression of AQPs 3 and 9 in the amnion. A tritiated glycerol test enabled us to measure the impact of insulin on the functional characteristics. Using an inhibitor of phosphatidylinositol 3-kinase, we analyzed the insulin intracellular signaling pathway. Results: The expression of AQP3 protein was significantly weaker in groups T2D and GD. In nondiabetic fetal membranes, we showed for the amnion (but not for the chorion) a significant repression by insulin of the transcriptional expression of AQPs 3 and 9, which was blocked by a phosphatidylinositol 3-kinase inhibitor. Conclusion: In fetal membranes, the repression of AQP3 protein expression and functions observed in vivo is allowed by the hyperinsulinism described in pregnant women with T2D or GD.

scholarly journals Correction of RNA splicing defect in β654-thalassemia mice using CRISPR/Cas9 gene-editing technology

Haematologica ◽  
2021 ◽  
Author(s):  
Dan Lu ◽  
Xiuli Gong ◽  
Yudan Fang ◽  
Xinbing Guo ◽  
Yanwen Chen ◽  
...  
Keyword(s):  
Survival Rate ◽  
Genome Editing ◽  
Molecular Mechanism ◽  
Splice Site ◽  
Rna Splicing ◽  
Gene Editing ◽  
Clinical Symptoms ◽  
Rna Expression ◽  
Wild Type ◽  
Acceptor Splice Site

β654-thalassemia is a prominent Chinese subtype of β-thalassemia, representing 17% of total β-thalassemia cases in China. The molecular mechanism underlying this subtype involves the IVS-2-654 C→T mutation leading to aberrant β-globin RNA splicing. This results in an additional 73-nucleotide exon between exons 2 and 3 and leads to severe thalassemia syndrome. Herein, we explored a CRISPR/Cas9 genome editing approach to eliminate the additional 73-nt by targeting both the IVS-2-654 C→T and a cryptic acceptor splice site at IVS-2-579 in order to correct aberrant β-globin RNA splicing and ameliorate the clinical β-thalassemia syndrome in β654 mice. Gene-edited mice were generated by microinjection of sgRNAs and Cas9 mRNAs into 1-cell embryos of β654 or control mice. 83.3% of live-born mice were gene-edited, 70% of which produced correctly spliced RNA. No off-target events were observed. The clinical symptoms, including hematologic parameters and tissue pathology of all of the edited-β654 founders and their offspring, were significantly improved compared to the non-edited β654 mice, consistent with the restoration of wild-type β-globin RNA expression. Notably, the survival rate of gene-edited heterozygous β654 mice increased significantly, and live-born homozygous β654 mice were observed. Our study demonstrated a new and effective gene-editing approach that may provide a groundwork for the exploration of β654-thalassemia therapy in the future.

Type 1 diabetes mellitus associated with activated phosphatidylinositol 3-kinase delta syndrome, type 2

2018 ◽  
Vol 10 (5) ◽  
pp. 421-422
Author(s):  
Ryuichi Nakagawa ◽  
Kei Takasawa ◽  
Tzu-Wen Yeh ◽  
Kohsuke Imai ◽  
Kenichi Kashimada ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Type 1 Diabetes ◽  
Type 1 Diabetes Mellitus ◽  
Syndrome Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

scholarly journals Control of the Papillomavirus Early-to-Late Switch by Differentially Expressed SRp20

2008 ◽  
Vol 83 (1) ◽  
pp. 167-180 ◽  
Author(s):  
Rong Jia ◽  
Xuefeng Liu ◽  
Mingfang Tao ◽  
Michael Kruhlak ◽  
Ming Guo ◽  
...  
Keyword(s):  
Keratinocyte Differentiation ◽  
Bovine Papillomavirus ◽  
Cellular Transcription Factor ◽  
Monolayer Cultures ◽  
Papillomavirus Infection ◽  
Alternative Mrna Splicing ◽  
E6 And E7 ◽  
Cellular Transcription ◽  
Selection Of

ABSTRACT The viral early-to-late switch of papillomavirus infection is tightly linked to keratinocyte differentiation and is mediated in part by alternative mRNA splicing. Here, we report that SRp20, a cellular splicing factor, controls the early-to-late switch via interactions with A/C-rich RNA elements. An A/C-rich SE4 element regulates the selection of a bovine papillomavirus type 1 (BPV-1) late-specific splice site, and binding of SRp20 to SE4 suppresses this selection. Expression of late BPV-1 L1 or human papillomavirus (HPV) L1, the major capsid protein, inversely correlates with SRp20 levels in the terminally differentiated keratinocytes. In HPV type 16, a similar SRp20-interacting element also controls the viral early-to-late switch. Keratinocytes in raft cultures, which support L1 expression, make considerably less SRp20 than keratinocytes in monolayer cultures, which do not support L1 expression. Conversely, abundant SRp20 in cancer cells or undifferentiated keratinocytes is important for the expression of the viral early E6 and E7 by promoting the expression of cellular transcription factor SP1 for transactivation of viral early promoters.

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