scholarly journals Antisense Oligonucleotide-Mediated Splice Switching: Potential Therapeutic Approach for Cancer Mitigation

Cancers ◽  
2021 ◽  
Vol 13 (21) ◽  
pp. 5555
Author(s):  
Prithi Raguraman ◽  
Akilandeswari Ashwini Balachandran ◽  
Suxiang Chen ◽  
Sarah D. Diermeier ◽  
Rakesh N. Veedu
Keyword(s):  
Cancer Progression ◽  
Messenger Rna ◽  
Splice Variants ◽  
Stop Codon ◽  
Mrna Level ◽  
Premature Stop Codon ◽  
Exon Skipping ◽  
Protein Isoforms ◽  
Delivery Strategies ◽  
And Function

Splicing is an essential process wherein precursor messenger RNA (pre-mRNA) is reshaped into mature mRNA. In alternative splicing, exons of any pre-mRNA get rearranged to form mRNA variants and subsequently protein isoforms, which are distinct both by structure and function. On the other hand, aberrant splicing is the cause of many disorders, including cancer. In the past few decades, developments in the understanding of the underlying biological basis for cancer progression and therapeutic resistance have identified many oncogenes as well as carcinogenic splice variants of essential genes. These transcripts are involved in various cellular processes, such as apoptosis, cell signaling and proliferation. Strategies to inhibit these carcinogenic isoforms at the mRNA level are promising. Antisense oligonucleotides (AOs) have been developed to inhibit the production of alternatively spliced carcinogenic isoforms through splice modulation or mRNA degradation. AOs can also be used to induce splice switching, where the expression of an oncogenic protein can be inhibited by the induction of a premature stop codon. In general, AOs are modified chemically to increase their stability and binding affinity. One of the major concerns with AOs is efficient delivery. Strategies for the delivery of AOs are constantly being evolved to facilitate the entry of AOs into cells. In this review, the different chemical modifications employed and delivery strategies applied are discussed. In addition to that various AOs in clinical trials and their efficacy are discussed herein with a focus on six distinct studies that use AO-mediated exon skipping as a therapeutic strategy to combat cancer.

scholarly journals Interferon lambda 4 expression is suppressed by the host during viral infection

2016 ◽  
Vol 213 (12) ◽  
pp. 2539-2552 ◽  
Author(s):  
MeeAe Hong ◽  
Johannes Schwerk ◽  
Chrissie Lim ◽  
Alison Kell ◽  
Abigail Jarret ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Splice Variants ◽  
Stop Codon ◽  
Association Studies ◽  
Premature Stop Codon ◽  
Genetic Association Studies ◽  
Protein Isoforms ◽  
Translation Efficiency ◽  
Regulation Of Expression ◽  
Protein Coding

Interferon (IFN) lambdas are critical antiviral effectors in hepatic and mucosal infections. Although IFNλ1, IFNλ2, and IFNλ3 act antiviral, genetic association studies have shown that expression of the recently discovered IFNL4 is detrimental to hepatitis C virus (HCV) infection through a yet unknown mechanism. Intriguingly, human IFNL4 harbors a genetic variant that introduces a premature stop codon. We performed a molecular and biochemical characterization of IFNλ4 to determine its role and regulation of expression. We found that IFNλ4 exhibits similar antiviral activity to IFNλ3 without negatively affecting antiviral IFN activity or cell survival. We show that humans deploy several mechanisms to limit expression of functional IFNλ4 through noncoding splice variants and nonfunctional protein isoforms. Furthermore, protein-coding IFNL4 mRNA are not loaded onto polyribosomes and lack a strong polyadenylation signal, resulting in poor translation efficiency. This study provides mechanistic evidence that humans suppress IFNλ4 expression, suggesting that immune function is dependent on other IFNL family members.

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2501-2505 ◽  
Author(s):  
Maurizio Margaglione ◽  
Rosa Santacroce ◽  
Donatella Colaizzo ◽  
Davide Seripa ◽  
Gennaro Vecchione ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Autosomal Recessive Disorder ◽  
Chain Gene ◽  
Hemorrhagic Diathesis ◽  
Reading Frame ◽  
Congenital Afibrinogenemia

Abstract Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

scholarly journals Alternative REST Splicing Underappreciated

2017 ◽  
Author(s):  
Guo-Lin Chen ◽  
Gregory M. Miller
Keyword(s):  
Alternative Splicing ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Protein Isoforms ◽  
Multiple Protein ◽  
Mrna Variants ◽  
Cellular Context ◽  
Active Transcription ◽  
Context Dependent ◽  
Ageing Brain

As a major orchestrator of the cellular epigenome, the repressor element-1 silencing transcription factor (REST) can either repress or activate thousands of genes depending on cellular context, suggesting a highly context-dependent REST function tuned by environmental cues. While REST shows cell-type non-selective active transcription1, an N-terminal REST4 isoform caused by alternative splicing – inclusion of an extra exon (N3c) which introduces a premature stop codon – has been implicated in neurogenesis and tumorigenesis2-5. Recently, in line with established epigenetic regulation of pre-mRNA splicing6,7, we demonstrated that REST undergoes extensive, context-dependent alternative splicing which results in the formation of a large number of mRNA variants predictive of multiple protein isoforms8. Supported by that immunoblotting/-staining with different anti-REST antibodies yield inconsistent results, alternative splicing allows production of various structurally and functionally different REST protein isoforms in response to shifting physiological requirements, providing a reasonable explanation for the diverse, highly context-dependent REST function. However, REST isoforms might be differentially assayed or manipulated, leading to data misinterpretation and controversial findings. For example, in contrast to the proposed neurotoxicity of elevated nuclear REST in ischemia9 and Huntington’s disease10,11, Lu et al. recently reported decreased nuclear REST in Alzheimer’s disease and neuroprotection of REST in ageing brain12. Unfortunately, alternative REST splicing was largely neglected by Lu et al., making it necessary for a reevaluation of their findings.

scholarly journals Restoring Dystrophin Expression in Duchenne Muscular Dystrophy: Current Status of Therapeutic Approaches

2019 ◽  
Vol 9 (1) ◽  
pp. 1 ◽  
Author(s):  
Yuko Shimizu-Motohashi ◽  
Hirofumi Komaki ◽  
Norio Motohashi ◽  
Shin’ichi Takeda ◽  
Toshifumi Yokota ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Stop Codon ◽  
Genetic Disorder ◽  
Premature Stop Codon ◽  
Exon Skipping ◽  
Current Status ◽  
Therapeutic Approaches ◽  
Dystrophin Expression

Duchenne muscular dystrophy (DMD), a rare genetic disorder characterized by progressive muscle weakness, is caused by the absence or a decreased amount of the muscle cytoskeletal protein dystrophin. Currently, several therapeutic approaches to cure DMD are being investigated, which can be categorized into two groups: therapies that aim to restore dystrophin expression, and those that aim to compensate for the lack of dystrophin. Therapies that restore dystrophin expression include read-through therapy, exon skipping, vector-mediated gene therapy, and cell therapy. Of these approaches, the most advanced are the read-through and exon skipping therapies. In 2014, ataluren, a drug that can promote ribosomal read-through of mRNA containing a premature stop codon, was conditionally approved in Europe. In 2016, eteplirsen, a morpholino-based chemical capable of skipping exon 51 in premature mRNA, received conditional approval in the USA. Clinical trials on vector-mediated gene therapy carrying micro- and mini- dystrophin are underway. More innovative therapeutic approaches include CRISPR/Cas9-based genome editing and stem cell-based cell therapies. Here we review the current status of therapeutic approaches for DMD, focusing on therapeutic approaches that can restore dystrophin.

scholarly journals Revised Exon Structure of l-DOPA Decarboxylase (DDC) Reveals Novel Splice Variants Associated with Colorectal Cancer Progression

2020 ◽  
Vol 21 (22) ◽  
pp. 8568
Author(s):  
Pinelopi I. Artemaki ◽  
Maria Papatsirou ◽  
Michaela A. Boti ◽  
Panagiotis G. Adamopoulos ◽  
Spyridon Christodoulou ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Progression ◽  
Prognostic Value ◽  
Splice Variants ◽  
Protein Isoforms ◽  
Dopa Decarboxylase ◽  
Colorectal Cancer Progression ◽  
Next Generation Sequencing Ngs ◽  
Exon Structure

Colorectal cancer (CRC) is a highly heterogenous malignancy with an increased mortality rate. Aberrant splicing is a typical characteristic of CRC, and several studies support the prognostic value of particular transcripts in this malignancy. l-DOPA decarboxylase (DDC) and its derivative neurotransmitters play a multifaceted role in physiological and pathological states. Our recent data support the existence of 6 DDC novel exons. In this study, we investigated the existence of additional DDC novel exons and transcripts, and their potential value as biomarkers in CRC. Next-generation sequencing (NGS) in 55 human cell lines coupled with Sanger sequencing uncovered 3 additional DDC novel exons and 20 splice variants, 7 of which likely encode new protein isoforms. Eight of these transcripts were detected in CRC. An in-house qPCR assay was developed and performed in TNM II and III CRC samples for the quantification of transcripts bearing novel exons. Extensive biostatistical analysis uncovered the prognostic value of specific DDC novel exons for patients’ disease-free and overall survival. The revised DDC exon structure, the putative protein isoforms with distinct functions, and the prognostic value of novel exons highlight the pivotal role of DDC in CRC progression, indicating its potential utility as a molecular biomarker in CRC.

scholarly journals Intronic splicing of hyaluronan synthase 1 (HAS1): a biologically relevant indicator of poor outcome in multiple myeloma

Blood ◽  
2005 ◽  
Vol 105 (12) ◽  
pp. 4836-4844 ◽  
Author(s):  
Sophia Adamia ◽  
Tony Reiman ◽  
Mary Crainie ◽  
Michael J. Mant ◽  
Andrew R. Belch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Gammopathy ◽  
Splice Variants ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Hyaluronan Synthase ◽  
Gene Splicing ◽  
Biologically Relevant ◽  
Mitotic Abnormalities ◽  
The Family

Abstract In this study, we show that the hyaluronan synthase 1 (HAS1) gene undergoes aberrant intronic splicing in multiple myeloma (MM). In addition to HAS1 full length (HAS1FL), we identify 3 novel splice variants of HAS1, HAS1Va, HAS1Vb, and HAS1Vc, detected in patients with MM or monoclonal gammopathy of undetermined significance (MGUS). HAS1Vb and HAS1Vc undergo intronic splicing with creation of a premature stop codon. MM cells expressing one or more HAS1 variants synthesize extracellular and/or intracellular hyaluronan (HA). Expression of the HAS1Vb splice variant was significantly correlated with reduced survival (P = .001). Together, alternative HAS1 gene splicing, the correlations between HAS1 splicing and HA synthesis, and the correlations between HAS1 splicing and reduced survival of MM patients support the hypothesis that the family of HAS1 protein plays a significant role in disease progression. Further, expression of HAS1Vb, in conjunction with HAS1FL and/or other HAS1 variants, may lead to accumulation of intracellular HA molecules and an impact on receptor for HA-mediated motility (RHAMM)-mediated mitotic abnormalities in MM. This study highlights the potential importance of HAS1 and its alternative splicing in pathophysiology of MGUS and MM. (Blood. 2005;105: 4836-4844)

scholarly journals Mutations in the unc-52 gene responsible for body wall muscle defects in adult Caenorhabditis elegans are located in alternatively spliced exons.

Genetics ◽  
1995 ◽  
Vol 139 (1) ◽  
pp. 159-169 ◽  
Author(s):  
T M Rogalski ◽  
E J Gilchrist ◽  
G P Mullen ◽  
D G Moerman
Keyword(s):  
Caenorhabditis Elegans ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Exon Skipping ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Alternatively Spliced ◽  
Mutant Phenotypes ◽  
Alternatively Spliced Exons ◽  
Muscle Cell Membrane

Abstract The unc-52 gene in Caenorhabditis elegans produces several large proteins that function in the basement membrane underlying muscle cells. Mutations in this gene result in defects in myofilament assembly and in the attachment of the myofilament lattice to the muscle cell membrane. The st549 and ut111 alleles of unc-52 produce a lethal (Pat) terminal phenotype whereas the e444, e669, e998, e1012 and e1421 mutations result in viable, paralyzed animals. We have identified the sequence alterations responsible for these mutant phenotypes. The st549 allele has a premature stop codon in exon 7 that should result in the complete elimination of unc-52 gene function, and the ut111 allele has a Tc1 transposon inserted into the second exon of the gene. The five remaining mutations are clustered in a small interval containing three adjacent, alternatively spliced exons (16, 17 and 18). These mutations affect some, but not all of the unc-52-encoded proteins. Thirteen intragenic revertants of the e669, e998, e1012 and e1421 alleles have also been sequenced. The majority of these carry the original mutation plus a G to A transition in the conserved splice acceptor site of the affected exon. This result suggests that reversion of the mutant phenotype in these strains may be the result of exon-skipping.

scholarly journals A nonsense mutation in the 3-hydroxy-3-methylglutaryl-CoA lyase gene produces exon skipping in two patients of different origin with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency

1997 ◽  
Vol 323 (2) ◽  
pp. 329-335 ◽  
Author(s):  
Juan PIÉ ◽  
Núria CASALS ◽  
Cesar H. CASALE ◽  
Carlos BUESA ◽  
Cristina MASCARÓ ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nonsense Mutation ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Exon Skipping ◽  
Leader Peptide ◽  
Aberrant Splice ◽  
Splice Site Selection ◽  
Single Strand Conformational Polymorphism ◽  
Exon 2

A novel nonsense mutation associated with the skipping of constitutive exon 2 of the 3-hydroxy-3-methylglutaryl-CoA lyase gene was found in two patients, from Portugal and Morocco, with 3-hydroxy-3-methylglutaric acidemia. By reverse transcriptase PCR and single-strand conformational polymorphism a G–T transversion was located, at nucleotide 109, of the 3-hydroxy-3-methylglutaryl-CoA lyase cDNA, within exon 2. Two mRNAs were produced as a result of this nonsense mutation: one of the expected size that contains the premature stop codon UAA, and the other with a deletion of 84 bp corresponding to the whole of exon 2. This deletion produced the loss of the last seven amino acids of the leader peptide and the first 21 amino acids of the mature protein. The nonsense mutation was found in a purine-rich GGAAG sequence, which is equal to, or similar to, others reported to be exonic splicing enhancers (ESE). We suggest that the nonsense mutation may affect a possible ESE on exon 2, which would hinder the splice site selection and facilitate an aberrant splice with the skipping of this exon. Determination by quantitative PCR shows that the ratio of mRNA with the nonsense mutation to the mRNA with the deletion is approx. 3:1.

Characterization of Gene p306 Expressed in Monocyte Derived Dendritic Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2567-2567
Author(s):  
Ani ta Bringmann ◽  
Ingo Hilgendorf ◽  
Silke Appel ◽  
Stefanie Held ◽  
Karin von Schwarzenberg ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Protein Expression ◽  
Cellular Localization ◽  
Splice Variants ◽  
Mrna Level ◽  
Structural Features ◽  
Protein Isoforms ◽  
Poly I:C ◽  
Apoptosis Induction ◽  
New Genes

Abstract Dendritic cells (DCs) play a significant role in the initiation and maintenance of primary immune responses. The identification of new genes that are involved in DC biology is crucial for a better understanding of the unique DC functions. We approached this issue by generating a substractive cDNA library based on suppression hybridization between monocyte derived DCs (mDCs) cDNA and the reference monocyte cDNA. Among various differentially in mDC expressed genes, we identified the gene p306 with unknown functions. In the following, we characterized the gene p306 and the translated protein splice variants. We confirmed the p306 expression in mDCs on mRNA level by RACE and RT-PCR with subsequent sequencing. Besides the 2 known splice variants, we found a third novel form. Two splice variants were expressed in activated myeloid and plasmacytoid DCs sorted from blood. To characterize the p306 protein forms, we analyzed the hypothetical proteins by various prediction programs. We found that the N-terminus of the protein isoforms is homologue to a DNA-binding protein. Structural features are an oligonucleotide-binding fold with an integrated zinc ribbon. To detect p306 protein expression, we generated a polyclonal antibody and used it in Western blotting experiments. We were able to detect 3 protein bands with sizes of 36 kDa, 84 kDa and about 200 kDa, which differed from the expected sizes of 20 kDa and 111 kDa. We found that the unexpected sizes are in part due to N-glycosylation using the inhibitor tunicamycin. When analyzing cell lysates and nuclear extracts, we found that the 36 kDa protein form is expressed exclusively in the nucleus, while other forms could be detected in nucleus and cytosol. Interestingly, activation of mDCs with Toll like receptor ligands Pam3Cys, Poly I:C, LPS and R848 selectively upregulated the expression of the 84 kDa form in the nucleus. DC treatment with IL-10 or Imatinib, compounds that inhibit DC differentiation and function, abolished the expression of all 3 protein forms. In order to analyze the possible effect of proteases on the post-transcriptional regulation of the protein expression and generation of the identified splice variants, mDCs were treated with several protease inhibitors. DC treatment with cathepsin/subtilisin inhibitor led to the downregulation of all 3 protein variants, indicating that p306 might be involved in the regulation of apoptosis induction. The pan-caspase inhibitor zVAD caused the upregulation of all protein forms. In conclusion, we identified a novel gene p306 that is differentially expressed in mDC, which encodes for at least 3 different splice variants and protein forms with diverse cellular localization. Although the functions of the p306 proteins still have to be determined, the gene p306 is probably involved in binding nucleic acids and in regulating apoptosis induction.

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2501-2505
Author(s):  
Maurizio Margaglione ◽  
Rosa Santacroce ◽  
Donatella Colaizzo ◽  
Davide Seripa ◽  
Gennaro Vecchione ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Autosomal Recessive Disorder ◽  
Chain Gene ◽  
Hemorrhagic Diathesis ◽  
Reading Frame ◽  
Congenital Afibrinogenemia

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

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