scholarly journals Roles for SR Proteins and hnRNP A1 in the Regulation of c-src Exon N1

2003 ◽  
Vol 23 (6) ◽  
pp. 1874-1884 ◽  
Author(s):  
Nanette Rooke ◽  
Vadim Markovtsov ◽  
Esra Cagavi ◽  
Douglas L. Black
Keyword(s):  
Regulatory Element ◽  
Sr Proteins ◽  
Hnrnp A1 ◽  
Sr Protein ◽  
Enhancer Element ◽  
Splicing Regulators ◽  
In Vitro Splicing ◽  
Hnrnp F

ABSTRACT The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5′ half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.

scholarly journals Roles of hnRNP A1, SR Proteins, and p68 Helicase in c-H-ras Alternative Splicing Regulation

2003 ◽  
Vol 23 (8) ◽  
pp. 2927-2941 ◽  
Author(s):  
Sònia Guil ◽  
Renata Gattoni ◽  
Montserrat Carrascal ◽  
Joaquín Abián ◽  
James Stévenin ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Sr Proteins ◽  
Splicing Regulation ◽  
Hnrnp A1 ◽  
Carboxy Terminus ◽  
Extracellular Signals ◽  
Nuclear Extracts ◽  
Acting In Concert

ABSTRACT Human ras genes play central roles in coupling extracellular signals with complex intracellular networks controlling proliferation, differentiation, and apoptosis, among others processes. c-H-ras pre-mRNA can be alternatively processed into two mRNAs due to the inclusion or exclusion of the alternative exon IDX; this renders two proteins, p21H-Ras and p19H-RasIDX, which differ only at the carboxy terminus. Here, we have characterized some of the cis-acting sequences and trans-acting factors regulating IDX splicing. A downstream intronic silencer sequence (rasISS1), acting in concert with IDX, negatively regulates upstream intron splicing. This effect is mediated, at least in part, by the binding of hnRNP A1. Depletion and add-back experiments in nuclear extracts have confirmed hnRNP A1's inhibitory role in IDX splicing. Moreover, the addition of two SR proteins, SC35 and SRp40, can counteract this inhibition by strongly promoting the splicing of the upstream intron both in vivo and in vitro. Further, the RNA-dependent helicase p68 is also associated with both IDX and rasISS1 RNA, and suppression of p68 expression in HeLa cells by RNAi experiments results in a marked increase of IDX inclusion in the endogenous mRNA, suggesting a role for this protein in alternative splicing regulation.

scholarly journals Identification and Characterization of a Novel Serine-Arginine-Rich Splicing Regulatory Protein

2000 ◽  
Vol 20 (9) ◽  
pp. 3049-3057 ◽  
Author(s):  
Daron C. Barnard ◽  
James G. Patton
Keyword(s):  
Regulatory Protein ◽  
Structural Similarity ◽  
Sr Proteins ◽  
Rna Recognition Motif ◽  
Sr Protein ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Rs Domains

ABSTRACT We have identified an 86-kDa protein containing a single amino-terminal RNA recognition motif and two carboxy-terminal domains rich in serine-arginine (SR) dipeptides. Despite structural similarity to members of the SR protein family, p86 is clearly unique. It is not found in standard SR protein preparations, does not precipitate in the presence of high magnesium concentrations, is not recognized by antibodies specific for SR proteins, and cannot complement splicing-defective S100 extracts. However, we have found that p86 can inhibit the ability of purified SR proteins to activate splicing in S100 extracts and can even inhibit the in vitro and in vivo activation of specific splice sites by a subset of SR proteins, including ASF/SF2, SC35, and SRp55. In contrast, p86 activates splicing in the presence of SRp20. Thus, it appears that pairwise combination of p86 with specific SR proteins leads to altered splicing efficiency and differential splice site selection. In all cases, such regulation requires the presence of the two RS domains and a unique intervening EK-rich region, which appear to mediate direct protein-protein contact between these family members. Full-length p86, but not a mutant lacking the RS-EK-RS domains, was found to preferentially interact with itself, SRp20, ASF/SF2, SRp55, and, to a slightly lesser extent, SC35. Because of the primary sequence and unique properties of p86, we have named this protein SRrp86 for SR-related protein of 86 kDa.

scholarly journals Phosphorylation Regulates In Vivo Interaction and Molecular Targeting of Serine/Arginine-rich Pre-mRNA Splicing Factors

1999 ◽  
Vol 145 (3) ◽  
pp. 447-455 ◽  
Author(s):  
Joanne M. Yeakley ◽  
Hélène Tronchère ◽  
James Olesen ◽  
Jacqueline A. Dyck ◽  
Huan-You Wang ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Sr Proteins ◽  
Molecular Targeting ◽  
Splicing Factors ◽  
Binding Studies ◽  
Sr Protein ◽  
Domain Interactions

The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain–mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein–specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain–mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein–specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Δ yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain–containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

scholarly journals The Protein Kinase Clk/Sty Directly Modulates SR Protein Activity: Both Hyper- and Hypophosphorylation Inhibit Splicing

1999 ◽  
Vol 19 (10) ◽  
pp. 6991-7000 ◽  
Author(s):  
Jayendra Prasad ◽  
Karen Colwill ◽  
Tony Pawson ◽  
James L. Manley
Keyword(s):  
Site Selection ◽  
Sr Proteins ◽  
Splicing Factors ◽  
Protein Splicing ◽  
Protein Activity ◽  
Splice Site Selection ◽  
Sr Protein ◽  
Constitutive Splicing

ABSTRACT The splicing of mammalian mRNA precursors requires both protein phosphorylation and dephosphorylation, likely involving modification of members of the SR protein family of splicing factors. Several kinases have been identified that can phosphorylate SR proteins in vitro, and transfection assays have provided evidence that at least one of these, Clk/Sty, can modulate splicing in vivo. But evidence that a specific kinase can directly affect the splicing activity of SR proteins has been lacking. Here, by using purified recombinant Clk/Sty, a catalytically inactive mutant, and individual SR proteins, we show that Clk/Sty directly affects the activity of SR proteins, but not other essential splicing factors, in reconstituted splicing assays. We also provide evidence that both hyper- and hypophosphorylation inhibit SR protein splicing activity, repressing constitutive splicing and switching alternative splice site selection. These findings indicate that Clk/Sty directly and specifically influences the activity of SR protein splicing factors and, importantly, show that both under- and overphosphorylation of SR proteins can modulate splicing.

scholarly journals A Review on the Medicinal and Pharmacological Properties of Traditional Ethnomedicinal Plant Sonapatha, Oroxylum indicum

Sinusitis ◽  
2021 ◽  
Vol 5 (1) ◽  
pp. 71-89
Author(s):  
Ganesh Chandra Jagetia
Keyword(s):  
Glucose Transporter ◽  
Skin Diseases ◽  
Regulatory Element ◽  
Experimental Studies ◽  
Fatty Acid Synthetase ◽  
Preclinical Models ◽  
Oroxylin A ◽  
Oroxylum Indicum

Oroxylum indicum, Sonapatha is traditionally used to treat asthma, biliousness, bronchitis, diarrhea, dysentery, fevers, vomiting, inflammation, leukoderma, skin diseases, rheumatoid arthritis, wound injury, and deworm intestine. This review has been written by collecting the relevant information from published material on various ethnomedicinal and pharmacological aspects of Sonapatha by making an internet, PubMed, SciFinder, Science direct, and Google Scholar search. Various experimental studies have shown that Sonapatha scavenges different free radicals and possesses alkaloids, flavonoids, cardio glycosides, tannins, sterols, phenols, saponins, and other phytochemicals. Numerous active principles including oroxylin A, chrysin, scutellarin, baicalein, and many more have been isolated from the different parts of Sonapatha. Sonapatha acts against microbial infection, cancer, hepatic, gastrointestinal, cardiac, and diabetic disorders. It is useful in the treatment of obesity and wound healing in in vitro and in vivo preclinical models. Sonapatha elevates glutathione, glutathione-s-transferase, glutathione peroxidase, catalase, and superoxide dismutase levels and reduces aspartate transaminase alanine aminotransaminase, alkaline phosphatase, lactate dehydrogenase, and lipid peroxidation levels in various tissues. Sonapatha activates the expression of p53, pRb, Fas, FasL, IL-12, and caspases and inhibited nuclear factor kappa (NF-κB), cyclooxygenase (COX-2), tumor necrosis factor (TNFα), interleukin (IL6), P38 activated mitogen-activated protein kinases (MAPK), fatty acid synthetase (FAS), sterol regulatory element-binding proteins 1c (SREBP-1c), proliferator-activated receptor γ2 (PPARγ2), glucose transporter (GLUT4), leptin, and HPV18 oncoproteins E6 and E7 at the molecular level, which may be responsible for its medicinal properties. The phytoconstituents of Sonapatha including oroxylin A, chrysin, and baicalein inhibit the replication of SARS-CoV-2 (COVID-19) in in vitro and in vivo experimental models, indicating its potential to contain COVID-19 infection in humans. The experimental studies in various preclinical models validate the use of Sonapatha in ethnomedicine and Ayurveda.

scholarly journals RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing

Oncogene ◽  
2021 ◽  
Author(s):  
Qiuxia Yan ◽  
Peng Zeng ◽  
Xiuqin Zhou ◽  
Xiaoying Zhao ◽  
Runqiang Chen ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Aerobic Glycolysis ◽  
Histological Grade ◽  
Migration And Invasion ◽  
Binding Motif ◽  
Hnrnp A1

AbstractThe prognosis for patients with metastatic bladder cancer (BCa) is poor, and it is not improved by current treatments. RNA-binding motif protein X-linked (RBMX) are involved in the regulation of the malignant progression of various tumors. However, the role of RBMX in BCa tumorigenicity and progression remains unclear. In this study, we found that RBMX was significantly downregulated in BCa tissues, especially in muscle-invasive BCa tissues. RBMX expression was negatively correlated with tumor stage, histological grade and poor patient prognosis. Functional assays demonstrated that RBMX inhibited BCa cell proliferation, colony formation, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Mechanistic investigations revealed that hnRNP A1 was an RBMX-binding protein. RBMX competitively inhibited the combination of the RGG motif in hnRNP A1 and the sequences flanking PKM exon 9, leading to the formation of lower PKM2 and higher PKM1 levels, which attenuated the tumorigenicity and progression of BCa. Moreover, RBMX inhibited aerobic glycolysis through hnRNP A1-dependent PKM alternative splicing and counteracted the PKM2 overexpression-induced aggressive phenotype of the BCa cells. In conclusion, our findings indicate that RBMX suppresses BCa tumorigenicity and progression via an hnRNP A1-mediated PKM alternative splicing mechanism. RBMX may serve as a novel prognostic biomarker for clinical intervention in BCa.

Functional antagonism between YY1 and the serum response factor

1992 ◽  
Vol 12 (9) ◽  
pp. 4209-4214
Author(s):  
A Gualberto ◽  
D LePage ◽  
G Pons ◽  
S L Mader ◽  
K Park ◽  
...  
Keyword(s):  
Serum Response Factor ◽  
Regulatory Element ◽  
Target Sequence ◽  
Response Factor ◽  
Basal Expression ◽  
Actin Promoter ◽  
Alpha Actin ◽  
Serum Response

The rapid, transient induction of the c-fos proto-oncogene by serum growth factors is mediated by the serum response element (SRE). The SRE shares homology with the muscle regulatory element (MRE) of the skeletal alpha-actin promoter. It is not known how these elements respond to proliferative and cell-type-specific signals, but the response appears to involve the binding of the serum response factor (SRF) and other proteins. Here, we report that YY1, a multifunctional transcription factor, binds to SRE and MRE sequences in vitro. The methylation interference footprint of YY1 overlaps with that of the SRF, and YY1 competes with the SRF for binding to these DNA elements. Overexpression of YY1 repressed serum-inducible and basal expression from the c-fos promoter and repressed basal expression from the skeletal alpha-actin promoter. YY1 also repressed expression from the individual SRE and MRE sequences upstream from a TATA element. Unlike that of YY1, SRF overexpression alone did not influence the transcriptional activity of the target sequence, but SRF overexpression could reverse YY1-mediated trans repression. These data suggest that YY1 and the SRF have antagonistic functions in vivo.

scholarly journals Arabidopsis ADF5 Acts as a Downstream Target Gene of CBFs in Response to Low-Temperature Stress

2021 ◽  
Vol 9 ◽  
Author(s):  
Pan Zhang ◽  
Dong Qian ◽  
Changxin Luo ◽  
Yingzhi Niu ◽  
Tian Li ◽  
...  
Keyword(s):  
Low Temperature ◽  
Actin Cytoskeleton ◽  
Temperature Stress ◽  
Regulatory Element ◽  
Low Temperature Stress ◽  
Knockout Mutant ◽  
Triple Mutant ◽  
Freezing Resistance

Low temperature is a major adverse environment that affects normal plant growth. Previous reports showed that the actin cytoskeleton plays an important role in the plant response to low-temperature stress, but the regulatory mechanism of the actin cytoskeleton in this process is not clear. C-repeat binding factors (CBFs) are the key molecular switches for plants to adapt to cold stress. However, whether CBFs are involved in the regulation of the actin cytoskeleton has not been reported. We found that Arabidopsis actin depolymerizing factor 5 (ADF5), an ADF that evolved F-actin bundling function, was up-regulated at low temperatures. We also demonstrated that CBFs bound to the ADF5 promoter directly in vivo and in vitro. The cold-induced expression of ADF5 was significantly inhibited in the cbfs triple mutant. The freezing resistance of the adf5 knockout mutant was weaker than that of wild type (WT) with or without cold acclimation. After low-temperature treatment, the actin cytoskeleton of WT was relatively stable, but the actin cytoskeletons of adf5, cbfs, and adf5 cbfs were disturbed to varying degrees. Compared to WT, the endocytosis rate of the amphiphilic styryl dye FM4-64 in adf5, cbfs, and adf5 cbfs at low temperature was significantly reduced. In conclusion, CBFs directly combine with the CRT/DRE DNA regulatory element of the ADF5 promoter after low-temperature stress to transcriptionally activate the expression of ADF5; ADF5 further regulates the actin cytoskeleton dynamics to participate in the regulation of plant adaptation to a low-temperature environment.

Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements

Development ◽  
1998 ◽  
Vol 125 (22) ◽  
pp. 4349-4358 ◽  
Author(s):  
J. Charite ◽  
W. de Graaff ◽  
D. Consten ◽  
M.J. Reijnen ◽  
J. Korving ◽  
...  
Keyword(s):  
Binding Sites ◽  
Hox Genes ◽  
Regulatory Element ◽  
Ectopic Expression ◽  
Positional Information ◽  
Regulatory Elements ◽  
Gene Products ◽  
Anteroposterior Patterning

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

Drosophila transcriptional repressor protein that binds specifically to negative control elements in fat body enhancers

1992 ◽  
Vol 12 (9) ◽  
pp. 4093-4103
Author(s):  
D Falb ◽  
T Maniatis
Keyword(s):  
Binding Site ◽  
Fat Body ◽  
Regulatory Element ◽  
Negative Control ◽  
Nuclear Extracts ◽  
Eukaryotic Proteins ◽  
Adh Gene ◽  
A Site

Expression of the Drosophila melanogaster Adh gene in adults requires a fat body-specific enhancer called the Adh adult enhancer (AAE). We have identified a protein in Drosophila nuclear extracts that binds specifically to a site within the AAE (adult enhancer factor 1 [AEF-1]). In addition, we have shown that AEF-1 binds specifically to two other Drosophila fat body enhancers. Base substitutions in the AEF-1 binding site that disrupt AEF-1 binding in vitro result in a significant increase in the level of Adh expression in vivo. Thus, the AEF-1 binding site is a negative regulatory element within the AAE. A cDNA encoding the AEF-1 protein was isolated and shown to act as a repressor of the AAE in cotransfection studies. The AEF-1 protein contains four zinc fingers and an alanine-rich sequence. The latter motif is found in other eukaryotic proteins known to be transcriptional repressors.

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