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1.
Cancers (Basel) ; 14(8)2022 Apr 14.
Article in English | MEDLINE | ID: mdl-35454897

ABSTRACT

Alternative splicing (AS) is a procedure during gene expression that allows the production of multiple mRNAs from a single gene, leading to a larger number of proteins with various functions. The alternative splicing (AS) of Fas (Apo-1/CD95) pre-mRNA can generate membrane-bound or soluble isoforms with pro-apoptotic and anti-apoptotic functions. SRSF6, a member of the Serine/Arginine-rich protein family, plays essential roles in both constitutive and alternative splicing. Here, we identified SRSF6 as an important regulatory protein in Fas AS. The cassette exon inclusion of Fas was decreased by SRSF6-targeting shRNA treatment, but increased by SRSF6 overexpression. The deletion and substitution mutagenesis of the Fas minigene demonstrated that the UGCCAA sequence in the cassette exon of the Fas gene causes the functional disruption of SRSF6, indicating that these sequences are essential for SRSF6 function in Fas splicing. In addition, biotin-labeled RNA-pulldown and immunoblotting analysis showed that SRSF6 interacted with these RNA sequences. Mutagenesis in the splice-site strength alteration demonstrated that the 5' splice-site, but not the 3' splice-site, was required for the SRSF6 regulation of Fas pre-mRNA. In addition, a large-scale RNA-seq analysis using GTEX and TCGA indicated that while SRSF6 expression was correlated with Fas expression in normal tissues, the correlation was disrupted in tumors. Furthermore, high SRSF6 expression was linked to the high expression of pro-apoptotic and immune activation genes. Therefore, we identified a novel RNA target with 5' splice-site dependence of SRSF6 in Fas pre-mRNA splicing, and a correlation between SRSF6 and Fas expression.

2.
Cancers (Basel) ; 13(12)2021 Jun 20.
Article in English | MEDLINE | ID: mdl-34202984

ABSTRACT

Breast cancer is the most frequently occurred cancer type and the second cause of death in women worldwide. Alternative splicing (AS) is the process that generates more than one mRNA isoform from a single gene, and it plays a major role in expanding the human protein diversity. Aberrant AS contributes to breast cancer metastasis and resistance to chemotherapeutic interventions. Therefore, identifying cancer-specific isoforms is the prerequisite for therapeutic interventions intended to correct aberrantly expressed AS events. Here, we performed RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq) in breast cancer cells, to identify global breast cancer-specific AS defects. By RT-PCR validation, we demonstrate the high accuracy of RASL-seq results. In addition, we analyzed identified AS events using the Cancer Genome Atlas (TCGA) database in a large number of non-pathological and breast tumor specimens and validated them in normal and breast cancer samples. Interestingly, aberrantly regulated AS cassette exons in cancer tissues do not encode for known functional domains but instead encode for amino acids constituting regions of intrinsically disordered protein portions characterized by high flexibility and prone to be subjected to post-translational modifications. Collectively, our results reveal novel AS errors occurring in human breast cancer, potentially affecting breast cancer-related biological processes.

3.
Cells ; 10(3)2021 03 19.
Article in English | MEDLINE | ID: mdl-33808656

ABSTRACT

Alternative splicing (AS) is an important posttranscriptional regulatory process. Damaged or unnecessary cells need to be removed though apoptosis to maintain physiological processes. Caspase-2 pre-mRNA produces pro-apoptotic long mRNA and anti-apoptotic short mRNA isoforms through AS. How AS of Caspase-2 is regulated remains unclear. In the present study, we identified a novel regulatory protein SRSF9 for AS of Caspase-2 cassette exon 9. Knock-down (KD) of SRSF9 increased inclusion of cassette exon and on the other hand, overexpression of SRSF9 decreased inclusion of this exon. Deletion mutagenesis demonstrated that exon 9, parts of intron 9, exon 8 and exon 10 were not required for the role of SRSF9 in Caspase-2 AS. However, deletion and substitution mutation analysis revealed that AGGAG sequence located at exon 10 provided functional target for SRSF9. In addition, RNA-pulldown mediated immunoblotting analysis showed that SRSF9 interacted with this sequence. Gene ontology analysis of RNA-seq from SRSF9 KD cells demonstrates that SRSF9 could regulate AS of a subset of apoptosis related genes. Collectively, our results reveal a basis for regulation of Caspase-2 AS.


Subject(s)
Caspase 2/metabolism , Exons/genetics , Serine-Arginine Splicing Factors/metabolism , Caspase 2/genetics , Cell Line, Tumor , Humans , RNA Precursors/genetics , RNA Splicing/physiology , RNA-Binding Proteins/metabolism , Serine-Arginine Splicing Factors/genetics , Transcription Factors/metabolism
4.
Cells ; 10(4)2021 04 09.
Article in English | MEDLINE | ID: mdl-33918758

ABSTRACT

Aberrant alternative splicing (AS) is a hallmark of cancer and a potential target for novel anti-cancer therapeutics. Breast cancer-associated AS events are known to be linked to disease progression, metastasis, and survival of breast cancer patients. To identify altered AS programs occurring in metastatic breast cancer, we perform a global analysis of AS events by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq). We demonstrate that, relative to low-metastatic, high-metastatic breast cancer cells show different AS choices in genes related to cancer progression. Supporting a global reshape of cancer-related splicing profiles in metastatic breast cancer we found an enrichment of RNA-binding motifs recognized by several splicing regulators, which have aberrant expression levels or activity during breast cancer progression, including SRSF1. Among SRSF1-regulated targets we found DCUN1D5, a gene for which skipping of exon 4 in its pre-mRNA introduces a premature termination codon (PTC), thus generating an unstable transcript degraded by nonsense-mediated mRNA decay (NMD). Significantly, distinct breast cancer subtypes show different DCUN1D5 isoform ratios with metastatic breast cancer expressing the highest level of the NMD-insensitive DCUN1D5 mRNA, thus showing high DCUN1D5 expression levels, which are ultimately associated with poor overall and relapse-free survival in breast cancer patients. Collectively, our results reveal global AS features of metastatic breast tumors, which open new possibilities for the treatment of these aggressive tumor types.


Subject(s)
Alternative Splicing/genetics , Breast Neoplasms/genetics , Breast Neoplasms/secondary , Base Sequence , Cell Line, Tumor , Exons/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Nonsense Mediated mRNA Decay/genetics , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Peptide Synthases/genetics , Peptide Synthases/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , Serine-Arginine Splicing Factors/genetics , Serine-Arginine Splicing Factors/metabolism , Survival Analysis
5.
BMB Rep ; 54(3): 176-181, 2021 Mar.
Article in English | MEDLINE | ID: mdl-33050987

ABSTRACT

Bcl-x, a member of the Bcl-2 family, plays a key role in apoptosis. Alternative splicing of Bcl-x pre-mRNA through alternative 5' splice-site selection produces an anti-apoptotic mRNA isoform that includes exon 2b and a pro-apoptotic Bcl-x mRNA isoform that excludes exon 2b. Here we used Bcl-x minigene and identified SRSF2 and SRSF6 as two regulatory factors of 5' splice-site selection of Bcl-x pre-mRNA. We selected binding clusters closer to 5' splice-sites from multiple potential binding sites of SRSF2 and SRSF6 to perform loss of functions analysis through site-directed mutagenesis. Our results demonstrated that these mutations did not abolish regulatory functions of SRSF2 or SRSF6, indicating that a single binding motif or a cluster was not a functional target of these proteins in Bcl-x pre-mRNA splicing. Random deletion mutagenesis did not disrupt the role of SRSF2 and SRSF6. Importantly, mutagenesis of 5' splice-site to a conserved or a weaker score demonstrated that the weaker strength of the target 5' splice-site or higher strength of the other 5' splice-site strength limited the role of SRSF2 and SRSF6 in 5' splice-site activation. [BMB Reports 2021; 54(3): 176-181].


Subject(s)
Alternative Splicing/genetics , Phosphoproteins/metabolism , RNA Precursors/genetics , RNA Splice Sites/genetics , Serine-Arginine Splicing Factors/metabolism , bcl-X Protein/genetics , Cells, Cultured , HEK293 Cells , Humans , Mutation , Phosphoproteins/genetics , RNA Precursors/metabolism , Serine-Arginine Splicing Factors/genetics , bcl-X Protein/metabolism
6.
Cells ; 9(12)2020 12 09.
Article in English | MEDLINE | ID: mdl-33317029

ABSTRACT

Splicing factor 3b subunit 1 (SF3B1) is an essential protein in spliceosomes and mutated frequently in many cancers. While roles of SF3B1 in single intron splicing and roles of its cancer-linked mutant in aberrant splicing have been identified to some extent, regulatory functions of wild-type SF3B1 in alternative splicing (AS) are not well-understood yet. Here, we applied RNA sequencing (RNA-seq) to analyze genome-wide AS in SF3B1 knockdown (KD) cells and to identify a large number of skipped exons (SEs), with a considerable number of alternative 5' splice-site selection, alternative 3' splice-site selection, mutually exclusive exons (MXE), and retention of introns (RI). Among altered SEs by SF3B1 KD, survival motor neuron 2 (SMN2) pre-mRNA exon 7 splicing was a regulatory target of SF3B1. RT-PCR analysis of SMN exon 7 splicing in SF3B1 KD or overexpressed HCT116, SH-SY5Y, HEK293T, and spinal muscular atrophy (SMA) patient cells validated the results. A deletion mutation demonstrated that the U2 snRNP auxiliary factor 65 kDa (U2AF65) interaction domain of SF3B1 was required for its function in SMN exon 7 splicing. In addition, mutations to lower the score of the polypyrimidine tract (PPT) of exon 7, resulting in lower affinity for U2AF65, were not able to support SF3B1 function, suggesting the importance of U2AF65 in SF3B1 function. Furthermore, the PPT of exon 7 with higher affinity to U2AF65 than exon 8 showed significantly stronger interactions with SF3B1. Collectively, our results revealed SF3B1 function in SMN alternative splicing.


Subject(s)
Alternative Splicing , Phosphoproteins/metabolism , RNA Splicing Factors/metabolism , Splicing Factor U2AF/metabolism , Survival of Motor Neuron 1 Protein/genetics , Survival of Motor Neuron 2 Protein/genetics , Cell Line , Exons , Humans , Muscular Atrophy, Spinal/metabolism , Muscular Atrophy, Spinal/pathology , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Polypyrimidine Tract-Binding Protein/genetics , Polypyrimidine Tract-Binding Protein/metabolism , Protein Binding , RNA Interference , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing Factors/antagonists & inhibitors , RNA Splicing Factors/genetics , RNA, Small Interfering/metabolism , Splicing Factor U2AF/chemistry , Survival of Motor Neuron 1 Protein/metabolism , Survival of Motor Neuron 2 Protein/metabolism
7.
Cancers (Basel) ; 12(11)2020 Oct 30.
Article in English | MEDLINE | ID: mdl-33143085

ABSTRACT

CD44 is a transmembrane glycoprotein involved in cell-cell and cell-matrix interactions. Several CD44 protein isoforms are generated in human through alternative splicing regulation of nine variable exons encoding for the extracellular juxta-membrane region. While the CD44 splicing variants have been described to be involved in cancer progression and development, the regulatory mechanism(s) underlying their production remain unclear. Here, we identify Tra2ß and SRSF9 as proteins with opposite roles in regulating CD44 exon v10 splicing. While Tra2ß promotes v10 inclusion, SRSF9 inhibits its inclusion. Mechanistically, we found that both proteins are able to target v10 exon, with GAAGAAG sequence being the binding site for Tra2ß and AAGAC that for SRSF9. Collectively, our data add a novel layer of complexity to the sequential series of events involved in the regulation of CD44 splicing.

8.
Cells ; 9(4)2020 04 10.
Article in English | MEDLINE | ID: mdl-32290247

ABSTRACT

The ratio control of 4R-Tau/3R-Tau by alternative splicing of Tau exon 10 is important for maintaining brain functions. In this study, we show that hnRNP A1 knockdown induces inclusion of endogenous Tau exon 10, conversely, overexpression of hnRNP A1 promotes exon 10 skipping of Tau. In addition, hnRNP A1 inhibits splicing of intron 9, but not intron 10. Furthermore, hnRNP A1 directly interacts with the 3' splice site of exon 10 to regulate its functions in alternative splicing. Finally, gene ontology analysis demonstrates that hnRNP A1-induced splicing and gene expression targets a subset of genes with neuronal function.


Subject(s)
Alternative Splicing/genetics , Exons/genetics , Heterogeneous Nuclear Ribonucleoprotein A1/genetics , RNA Splice Sites/genetics , tau Proteins/genetics , Humans , Transfection
9.
BMB Rep ; 52(11): 641-646, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31401978

ABSTRACT

The Ron proto-oncogene is a human receptor for macrophage-stimulating protein (MSP). The exclusion of exon 11 in alternative splicing generates ΔRON protein that is constitutively activated. Heterogenous ribonucleaoprotein (hnRNP) C1/C2 is one of the most abundant proteins in cells. In this manuscript, we showed that both hnRNP C1 and C2 promoted exon 11 inclusion of Ron pre-mRNA and that hnRNP C1 and hnRNP C2 functioned independently but not cooperatively. Moreover, hnRNP C1 stimulated exon 11 splicing through intron 10 activation but not through intron 11 splicing. Furthermore, we showed that, whereas the RRM domain was required for hnRNP C1 function, the Asp/Glu domain was not. In conclusion, hnRNP C1/C2 promoted exon 11 splicing independently by stimulating intron 10 splicing through RRM but not through the Asp/Glu domain. [BMB Reports 2019; 52(11): 641-646].


Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein Group C/genetics , RNA Precursors/metabolism , RNA Recognition Motif/genetics , Alternative Splicing/genetics , Alternative Splicing/physiology , Exons/genetics , HEK293 Cells , HeLa Cells , Heterogeneous-Nuclear Ribonucleoprotein Group C/metabolism , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Introns/genetics , Nuclear Proteins/metabolism , Proto-Oncogene Mas , RNA Splicing , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism
10.
Cells ; 8(7)2019 07 10.
Article in English | MEDLINE | ID: mdl-31295920

ABSTRACT

Here we show that the serine/arginine rich splicing factor 2 (SRSF2) promotes cryptic 3' splice-site (3'AG') usage during cassette exon exclusion in survival of motor neuron (SMN2) minigenes. Deletion of the 3'AG' (3'AG'1), its associated branch point (BP') and polypyrimidine tract (PPT') sequences directs SRSF2 to promote a second 3'AG' (3'AG'2) with less conserved associated region for intron splicing. Furthermore, deletion of both 3'AG'1 and 3'AG'2 and their associated sequences triggered usage of a third 3'AG'3 that has very weak associated sequences. Interestingly, when intron splicing was directed to the 3'AG' cryptic splice-sites, intron splicing from the canonical 3'AG splice-site was reduced along with a decrease in cassette exon inclusion. Moreover, multiple SRSF2 binding sites within the intron are responsible for 3'AG' activation. We conclude that SRSF2 facilitates exon exclusion by activating a cryptic 3'AG' and inhibiting downstream intron splicing.


Subject(s)
Exons , Serine-Arginine Splicing Factors/metabolism , Alternative Splicing , Binding Sites , HEK293 Cells , Humans , Introns , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splice Sites , RNA Splicing , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Serine-Arginine Splicing Factors/genetics , Survival of Motor Neuron 2 Protein/genetics , Survival of Motor Neuron 2 Protein/metabolism
11.
Biochem Biophys Res Commun ; 506(3): 703-708, 2018 11 30.
Article in English | MEDLINE | ID: mdl-30376989

ABSTRACT

Alternative splicing of exon 6 in Fas pre-mRNA generates a membrane bound pro-apoptotic isoform or soluble anti-apoptotic isoform. SRSF4 is a member of Arginine-Serine rich (SR) protein family. Here we demonstrate that increased SRSF4 expression stimulates exon 6 inclusion, and that reduced SRSF4 expression promotes exon 6 exclusion. We also show that weaker but not stronger 5' splice-site strength of exon 6 abolishes the SRSF4 effects on exon 6 splicing. Furthermore, we identified a novel enhancer on exon 6, on which SRSF4 interacts functionally and physically. Our results illustrate a novel regulatory mechanism of Fas pre-mRNA splicing.


Subject(s)
Enhancer Elements, Genetic/genetics , Exons/genetics , RNA Precursors/metabolism , RNA Splicing/genetics , Serine-Arginine Splicing Factors/metabolism , fas Receptor/genetics , Base Sequence , Gene Expression Regulation , HCT116 Cells , Humans , Protein Binding/genetics , RNA Precursors/genetics , RNA Splice Sites/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , fas Receptor/metabolism
12.
BMB Rep ; 50(8): 423-428, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28712387

ABSTRACT

SRSF2, a Serine-Arginine rich (SR) protein, is a splicing activator that mediates exon inclusion and exclusion events equally well. Here we show SRSF2 directly suppresses intron splicing to suppress cassette exon inclusion in SMN premRNA. Through a serial mutagenesis, we demonstrate that a 10 nt RNA sequence surrounding the branch-point (BP), is important for SRSF2-mediated inhibition of cassette exon inclusion through directly interacting with SRSF2. We conclude that SRSF2 inhibits intron splicing to promote exon exclusion. [BMB Reports 2017; 50(8): 423-428].


Subject(s)
Exons , Introns , Serine-Arginine Splicing Factors/genetics , Serine-Arginine Splicing Factors/metabolism , Alternative Splicing , Base Sequence , HEK293 Cells , Humans , Mutagenesis, Insertional , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing , RNA Splicing Factors/metabolism , Regulatory Elements, Transcriptional , SMN Complex Proteins/metabolism
13.
Oncol Lett ; 13(3): 1944-1948, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28454348

ABSTRACT

The récepteur d'origine nantais (RON) gene is a proto-oncogene that is responsible for encoding the human macrophage-stimulating protein (MSP) 1 receptor. MSP activation induces RON-mediated cell dissociation, migration and matrix invasion. Isoforms of RON that exclude exons 5 and 6 encode the RONΔ160 protein, which promotes cell transformation in vitro and tumor metastasis in vivo. Premature termination codons (PTCs) in exons activate the nonsense-mediated mRNA decay (NMD) signaling pathway. The present study demonstrated that PTCs at various locations in the alternative exons 5 and 6 could induce NMD of the majority of the spliced, or partially spliced, isoforms. However, the isoforms that excluded exon 6 or exons 5 and 6 were markedly increased when produced from mutated minigenes with inserted PTCs. Furthermore, the unspliced isoform of intron 5 was not observed to be decreased by the presence of PTCs. Notably, these effects may be dependent on the location of the PTCs. The current study demonstrated a novel mechanism underlying the regulation of NMD in alternative splicing.

14.
Biochim Biophys Acta Gene Regul Mech ; 1860(3): 363-373, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28119102

ABSTRACT

Selection of 5' splice-sites (5'SS) in alternative splicing plays an important role in gene regulation. Although regulatory mechanisms of heterogeneous nuclear ribonucleoprotein L (hnRNP L), a well-known splicing regulatory protein, have been studied in a substantial level, its role in 5'SS selection is not thoroughly defined. By using a KLF6 pre-mRNA alternative splicing model, we demonstrate in this report that hnRNP L inhibits proximal 5'SS but promotes two consecutive distal 5'SS splicing, antagonizing SRSF1 roles in KLF6 pre-mRNA splicing. In addition, three consecutive CA-rich sequences in a CA cassette immediately upstream of the proximal 5'SS are all required for hnRNP L functions. Importantly, the CA-cassette locations on the proximal exon do not affect hnRNP L roles. We further show that the proximal 5'SS but not the two distal 5'SSs are essential for hnRNP L activities. Notably, in a Bcl-x pre-mRNA model that contains two alternative 5'SS but includes CA-rich elements at distal exon, we demonstrate that hnRNP L also suppresses nearby 5'SS activation. Taken together, we conclude that hnRNP L suppresses 5'SS selection through multiple exonic motifs.


Subject(s)
Exons , Nucleotide Motifs , RNA Precursors/metabolism , RNA Splice Sites/physiology , RNA Splicing/physiology , Ribonucleoproteins/metabolism , Cell Line, Tumor , Humans , Kruppel-Like Factor 6 , Kruppel-Like Transcription Factors/biosynthesis , Kruppel-Like Transcription Factors/genetics , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , RNA Precursors/genetics , Ribonucleoproteins/genetics , Serine-Arginine Splicing Factors/biosynthesis , Serine-Arginine Splicing Factors/genetics
15.
BMB Rep ; 50(1): 20-24, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27616359

ABSTRACT

Clustered regularly-interspaced short palindromic repeats (CRISPR) is a new and effective genetic editing tool. CRISPR was initially found in bacteria to protect it from virus invasions. In the first step, specific DNA strands of virus are identified by guide RNA that is composed of crRNA and tracrRNA. Then RNAse III is required for producing crRNA from pre-crRNA. In The second step, a crRNA:tracrRNA:Cas9 complex guides RNase III to cleave target DNA. After cleavage of DNA by CRISPR-Cas9, DNA can be fixed by Non- Homologous End Joining (NHEJ) and Homology Directed Repair (HDR). Whereas NHEJ is simple and random, HDR is much more complex and accurate. Gene editing by CRISPR is able to be applied to various biological field such as agriculture and treating genetic diseases in human. [BMB Reports 2017; 50(1): 20-24].


Subject(s)
CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing/methods , DNA/genetics , Humans , RNA Editing/genetics , RNA, Guide, Kinetoplastida/genetics
16.
BMB Rep ; 49(11): 612-616, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27530682

ABSTRACT

CD44 pre-mRNA includes 20 exons, of which exons 1-5 (C1-C5) and exons 16-20 (C6-C10) are constant exons, whereas exons 6-15 (V1-V10) are variant exons. V6-exon-containing isoforms have been known to be implicated in tumor cell invasion and metastasis. In the present study, we performed a SR protein screen for CD44 V6 splicing using overexpression and lentivirus-mediated shRNA treatment. Using a CD44 V6 minigene, we demonstrate that increased SRSF3 and SRSF4 expression do not affect V6 splicing, but increased expression of SRSF1, SRSF6 and SRSF9 significantly inhibit V6 splicing. In addition, using a constitutive exon-specific primer set, we could not detect alterations of CD44 splicing after SR protein-targeting shRNA treatment. However, using a V6 specific primer, we identified that reduced SRSF2 expression significantly reduced the V6 isoform, but increased V6-10 and V6,7-10 isoforms. Our results indicate that SR proteins are important regulatory proteins for CD44 V6 splicing. [BMB Reports 2016; 49(11): 612-616].


Subject(s)
Hyaluronan Receptors/genetics , RNA Precursors/metabolism , Serine-Arginine Splicing Factors/metabolism , Exons , Humans , Hyaluronan Receptors/metabolism , MCF-7 Cells , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Interference , RNA Splicing , RNA, Small Interfering/metabolism , Real-Time Polymerase Chain Reaction , Serine-Arginine Splicing Factors/antagonists & inhibitors , Serine-Arginine Splicing Factors/genetics
17.
Methods Mol Biol ; 1421: 35-44, 2016.
Article in English | MEDLINE | ID: mdl-26965255

ABSTRACT

RNA-protein interaction can be detected by RNA pull-down and immunoblotting methods. Here, we describe a method to detect RNA-protein interaction using RNA pull down and to identify the proteins that are pulled-down by the RNA using immunoblotting. In this protocol, RNAs with specific sequences are biotinylated and immobilized onto Streptavidin beads, which are then used to pull down interacting proteins from cellular extracts. The presence of a specific protein is subsequently verified by SDS- polyacrylamide gel electrophoresis and immunoblotting with antibodies. Interactions between the SMN RNA and the PSF protein and between the caspase-2 RNA and the SRSF3 protein (SRp20) in nuclear extract prepared from HeLa cells are illustrated as examples.


Subject(s)
Immunoblotting/methods , Proteins/metabolism , RNA/metabolism , Biotinylation , Electrophoresis, Polyacrylamide Gel/methods , HeLa Cells , Humans , Protein Binding , Proteins/analysis , RNA/analysis , Serine-Arginine Splicing Factors/analysis , Serine-Arginine Splicing Factors/metabolism , Streptavidin/metabolism
18.
J Cancer ; 6(12): 1346-51, 2015.
Article in English | MEDLINE | ID: mdl-26640595

ABSTRACT

RON receptor tyrosine kinase is a proto-oncogene that induces cell migration and matrix invasion. RONΔ160 protein, which is produced by exclusion of exon 5 and 6, promotes cell migration, matrix invasion and protection from apoptosis. Alternative splicing regulation of exon 5 and 6 is not well understood. In this manuscript, we identified several new RNA regulatory elements for alternative splicing of Ron proto-oncogene. Firstly, we demonstrated that RNA sequences from EcoRI cleavage sites regulate alternative splicing of Ron exon 5 and 6. Secondly, we showed that the ~30 nt RNA at upstream end of exon 4 and the ~33 nt RNA at downstream end of exon 7 also modulate splicing of exon 5 and 6. Thirdly, our results indicate that the RNA sequences of the ends in exon 4 and 7 are required for the regulatory functions of the RNA from restriction enzyme cleavage sites. Our results provide a new insight for regulation of alternative splicing of Ron proto-oncogene.

19.
Proc Natl Acad Sci U S A ; 112(32): 9926-31, 2015 Aug 11.
Article in English | MEDLINE | ID: mdl-26216990

ABSTRACT

U2 snRNP auxiliary factor 65 kDa (U2AF(65)) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF(65) stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (SMN) pre-mRNA. A stronger 5' splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF(65). U2AF(65) overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF(65) inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF(65) effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF(65) even inhibits general splicing of adenovirus major late (Ad ML) or ß-globin pre-mRNA. Thus, we conclude that U2AF(65) possesses a splicing Inhibitory function that leads to alternative exon skipping.


Subject(s)
Alternative Splicing/genetics , Exons/genetics , Nuclear Proteins/genetics , Ribonucleoproteins/genetics , Base Sequence , DNA-Binding Proteins/genetics , HEK293 Cells , Humans , Introns/genetics , Molecular Sequence Data , Nuclear Proteins/chemistry , Protein Binding , Protein Structure, Tertiary , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splice Sites/genetics , Ribonucleoproteins/chemistry , SMN Complex Proteins/genetics , Splicing Factor U2AF , Structure-Activity Relationship , Transcription Factors/genetics , Viral Proteins/genetics , beta-Globins/genetics , tau Proteins/genetics
20.
Oncol Rep ; 34(3): 1231-8, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26151392

ABSTRACT

CD44 is a transmembrane receptor for hyaluronic acid. CD44 pre-mRNA contains 19 exons, 9 of which are alternatively spliced. Among the CD44 spliced variants, the v4-7 variant, one of the v6 exon-containing isoforms that contains variable exon 4, 5, 6 and 7, confers metastatic potential to non-metastatic cells. Splicing of CD44 and the function of CD44 isoforms are different in breast cancer cells. hnRNP A1 is a ubiquitously expressed protein with an inhibitory function in pre-mRNA splicing. We showed that CD44v6 isoform, which includes all of the v6-containing mRNA isoforms, had the highest expression level in non-metatatic breast cancer cells (MCF7) when compared to the level in metastatic breast cancer cells (MDA-MB-231) and normal breast cells (MCF10A). Furthermore we showed that hnRNP A1 knockdown regulated splicing of CD44 differently in breast cancer cells. We showed here that CD44 isoform expression is completely different in MDA-MB-231 cells than that in MCF7 and MCF10A cells, whereas MCF7 and MCF10A cells had a similar expression pattern of CD44 isoforms. RT-PCR analysis of CD44v6 showed that MCF7 and MCF10A cells predominantly expressed the c5v6v7v8v9v10c6 isoform. However, in addition to this isoform, MDA-MB-231 cells also expressed the c5v6v8v9v10c6 and c5v6c6 isoforms. We also found that knockdown of hnRNP A1 significantly reduced the expression of c5v6v7v8v9v10c6 and c5v6v8v9v10c6, and promoted the expression of c5v6c6. hnRNP A1 knockdown significantly induced cell death. In addition, hnRNP A1 knockdown induced a decrease in cell invasion in the MDA-MB-231 cells. Our results indicate that the knockdown of hnRNP A1 has a specific function on the splicing of CD44 in breast cancer cells.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/biosynthesis , Hyaluronan Receptors/genetics , Neoplasm Invasiveness/genetics , Alternative Splicing , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cell Survival/genetics , Female , Fluorescent Antibody Technique , Gene Expression Regulation, Neoplastic/physiology , Gene Knockdown Techniques , Heterogeneous Nuclear Ribonucleoprotein A1 , Humans , Hyaluronan Receptors/biosynthesis , Immunoblotting , Neoplasm Invasiveness/pathology , Protein Isoforms , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , Transfection
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