Deoxynivalenol globally affects the selection of 3’ splice sites in human cells by suppressing the splicing factors, U2AF1 and SF1

Abstract Pre-mRNA splicing is an essential step for the expression of intron-containing genes, i.e., the majority of genes. Splicing is a high-fidelity process, as required for the correct expression of proteins. However, there is flexibility in the selection of competing splice sites, which gives rise to alternative splicing, a regulated process that greatly increases the diversity of the proteome. Splicing is catalyzed in a stepwise manner by the spliceosome, a macromolecular machine that consists of 5 small RNAs and more than 100 proteins. Key insights about the structure and dynamics of spliceosomal complexes have recently been obtained through cryo-electron microscopy studies. Dysregulated splicing can arise from mutations in the splice sites or regulatory elements of individual genes, from alterations in the levels of regulatory splicing factors (activators and repressors), or from mutations in splicing-factor genes. All of these scenarios can give rise to various cancers, depending on the affected gene and the cellular context. Interestingly, recurrent somatic heterozygous mutations in particular splicing factors involved in branchpoint-sequence and 3'-splice-site recognition have emerged as key drivers of certain myeloid neoplasias. This presentation will review relevant features of the spliceosome machinery, the functional implications for normal and pathological conditions, and the potential for novel therapies. Disclosures Krainer: Ionis Pharmaceuticals: Consultancy, Honoraria, Patents & Royalties, Research Funding; Stoke Therapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, Patents & Royalties; Cold Spring Harbor Laboratory: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; H3 Biomedicine: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Structural disruption of exonic stem–loops immediately upstream of the intron regulates mammalian splicing

Nucleic Acids Research ◽

10.1093/nar/gkaa358 ◽

2020 ◽

Vol 48 (11) ◽

pp. 6294-6309 ◽

Cited By ~ 2

Author(s):

Kaushik Saha ◽

Whitney England ◽

Mike Minh Fernandez ◽

Tapan Biswas ◽

Robert C Spitale ◽

...

Keyword(s):

Structural Information ◽

Structural Features ◽

Splicing Factors ◽

Splice Sites ◽

Exonic Splicing Enhancer ◽

Stem Loop ◽

The Core ◽

Retained Introns ◽

Splicing Enhancer ◽

Structural Disruption

Abstract Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short (≲70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem–loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.

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Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells

BioMed Research International ◽

10.1155/2017/6037159 ◽

2017 ◽

Vol 2017 ◽

pp. 1-5 ◽

Cited By ~ 1

Author(s):

Hiroshi Sato ◽

Hiroki Kato ◽

Haruyoshi Yamaza ◽

Keiji Masuda ◽

Huong Thi Nguyen Nguyen ◽

...

Keyword(s):

Normal Cell ◽

Expression Vector ◽

Gene Dosage ◽

Genetic Disorders ◽

Human Cells ◽

Chromosome 21 ◽

Disease Phenotypes ◽

Simplex Virus ◽

Selection Of ◽

Trisomic Cell

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

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