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The Role of Alternative Pre-mRNA Spl...

Altieri, Jessie Anne Claire,

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The Role of Alternative Pre-mRNA Splicing in Biology and Disease /

Record Type:	Electronic resources : Monograph/item
Title/Author:	The Role of Alternative Pre-mRNA Splicing in Biology and Disease // Jessie Anne Claire Altieri.
Author:	Altieri, Jessie Anne Claire,
Description:	1 electronic resource (117 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 87-01, Section: B.
Contained By:	Dissertations Abstracts International87-01B.
Subject:	Biology. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=31848065
ISBN:	9798288802959

The Role of Alternative Pre-mRNA Splicing in Biology and Disease /
Altieri, Jessie Anne Claire,

The Role of Alternative Pre-mRNA Splicing in Biology and Disease /Jessie Anne Claire Altieri. - 1 electronic resource (117 pages)

Source: Dissertations Abstracts International, Volume: 87-01, Section: B.

Alternative pre-mRNA splicing generates protein diversity from a limited genome, yet its dysregulation contributes to various disease states. This dissertation presents four interconnected studies investigating the role of alternative splicing in normal cellular processes and human disease. The first project examined how 4-thiouridine (4sU), a widely used metabolic labeling tool, affects pre-mRNA splicing efficiency. Using in vitro splicing assays with varying 4sU incorporation levels and cell culture experiments, we demonstrated that elevated 4sU incorporation negatively correlates with splicing efficiency, particularly affecting introns with weaker splice sites. While standard labeling concentrations showed minimal effects on constitutive splicing, some alternative splicing events were significantly altered, highlighting the importance of appropriate controls in 4sU experiments.The second project investigated regulatory mechanisms governing exitron splicing, involving conditional removal of introns embedded within protein-coding exons. Using a Massively Parallel Reporter Assay with randomized sequences flanking a POLR3C exitron, we identified sequence features controlling splicing efficiency. Machine learning analysis achieved 83% accuracy in predicting splicing outcomes, with GC content as the dominant predictor, followed by uracil-rich pentamer motifs. Cross-referencing predictive motifs with RNA-binding protein databases revealed that heterogeneous nuclear ribonucleoproteins (hnRNPs) act as key splicing silencers.The third project explored pre-mRNA splicing in colorectal cancer. Building on findings that show dramatic increases in alternative splicing during colon stem cell differentiation, we hypothesized that stem-like splicing patterns associate with cancer development. Using murine intestinal organoids with defined genetic mutations (Apc-/- and Bmal1-/- ), we demonstrated that cancer-associated mutations lead to systematic splicing alterations. Key Wnt and proliferation genes, including KRAS, APC, and TCF7L2, showed cell-type-specific splicing signatures that were disrupted in cancer-predisposed organoids, which retained stem-like characteristics. The fourth project investigated a novel approach targeting SARS-CoV-2 infection by modulating ACE2 pre-mRNA splicing. We designed RNA antisense oligonucleotides (rASOs) targeting regulatory elements within ACE2 pre-mRNA to promote exon 17 skipping, altering the balance between membrane-bound and secreted ACE2 isoforms. Specific rASO combinations successfully induced exon 17 skipping, and functional assays revealed that supernatants from treated cells significantly reduced viral infectivity by over 50%. These studies advance our understanding of alternative splicing regulation and demonstrate its importance in normal cellular processes and disease states. The research provides methodological insights for RNA biology, identifies novel regulatory mechanisms controlling exitron splicing, establishes connections between splicing dysregulation and cancer, and demonstrates the potential of splicing modulation for antiviral applications.

English

ISBN: 9798288802959Subjects--Topical Terms:

522710
Biology.
Subjects--Index Terms:

Alternative splicing

The Role of Alternative Pre-mRNA Splicing in Biology and Disease /
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520 $a Alternative pre-mRNA splicing generates protein diversity from a limited genome, yet its dysregulation contributes to various disease states. This dissertation presents four interconnected studies investigating the role of alternative splicing in normal cellular processes and human disease. The first project examined how 4-thiouridine (4sU), a widely used metabolic labeling tool, affects pre-mRNA splicing efficiency. Using in vitro splicing assays with varying 4sU incorporation levels and cell culture experiments, we demonstrated that elevated 4sU incorporation negatively correlates with splicing efficiency, particularly affecting introns with weaker splice sites. While standard labeling concentrations showed minimal effects on constitutive splicing, some alternative splicing events were significantly altered, highlighting the importance of appropriate controls in 4sU experiments.The second project investigated regulatory mechanisms governing exitron splicing, involving conditional removal of introns embedded within protein-coding exons. Using a Massively Parallel Reporter Assay with randomized sequences flanking a POLR3C exitron, we identified sequence features controlling splicing efficiency. Machine learning analysis achieved 83% accuracy in predicting splicing outcomes, with GC content as the dominant predictor, followed by uracil-rich pentamer motifs. Cross-referencing predictive motifs with RNA-binding protein databases revealed that heterogeneous nuclear ribonucleoproteins (hnRNPs) act as key splicing silencers.The third project explored pre-mRNA splicing in colorectal cancer. Building on findings that show dramatic increases in alternative splicing during colon stem cell differentiation, we hypothesized that stem-like splicing patterns associate with cancer development. Using murine intestinal organoids with defined genetic mutations (Apc-/- and Bmal1-/- ), we demonstrated that cancer-associated mutations lead to systematic splicing alterations. Key Wnt and proliferation genes, including KRAS, APC, and TCF7L2, showed cell-type-specific splicing signatures that were disrupted in cancer-predisposed organoids, which retained stem-like characteristics. The fourth project investigated a novel approach targeting SARS-CoV-2 infection by modulating ACE2 pre-mRNA splicing. We designed RNA antisense oligonucleotides (rASOs) targeting regulatory elements within ACE2 pre-mRNA to promote exon 17 skipping, altering the balance between membrane-bound and secreted ACE2 isoforms. Specific rASO combinations successfully induced exon 17 skipping, and functional assays revealed that supernatants from treated cells significantly reduced viral infectivity by over 50%. These studies advance our understanding of alternative splicing regulation and demonstrate its importance in normal cellular processes and disease states. The research provides methodological insights for RNA biology, identifies novel regulatory mechanisms controlling exitron splicing, establishes connections between splicing dysregulation and cancer, and demonstrates the potential of splicing modulation for antiviral applications.
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650 4 $a Bioinformatics. $3 553671
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650 4 $a Oncology. $3 751006
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653 $a Pre-mRNA splicing
653 $a RNA antisense oligonucleotides
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