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2022 SHIJIE CUI PhD.pdf (4.59 MB)
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The mechanisms by which A1 regulates pre-mRNA splicing

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thesis
posted on 2023-09-13, 12:50 authored by Shijie Cui

In order to generate mRNA for proteins expression, pre-mRNA splicing is needed. In humans, 92–94% of genes undergo alternative splicing. Alternative splicing builds the diversity of gene expression and decides the expression level of gene products. The interaction of 𝘀π˜ͺ𝘴 -elements and trans-elements plays a major role in regulating alternative splicing. 𝘀π˜ͺ𝘴 -regulatory elements are sequences present in pre-mRNA, and trans-elements are splicing regulatory proteins, such as hnRNP proteins. HnRNP A1 (A1), which is mainly involved in alternative splicing events and plays an important role in splicing repression in opposition to SR proteins, includes an N-terminal domain that contains two RRMs and a C-terminal domain that is an arginine and glycine-rich protein-protein or protein-RNA interaction domain. A1 can also inhibit the binding of U1 snRNP to pre-mRNA to repress pre-mRNA splicing. SMN2 gene arose by an inversion of SMN1 gene. Due to the skipping of exon 7 in the splicing of SMN2, truncated SMN proteins are mainly. Accumulating evidences suggest that A1 plays an important role in regulating the splicing of SMN2. Our experiments with the established SMN2 RNA system in splicing have shown the significance of A1 motifs and the competition between A1 and U1 snRNP. Using single molecule microscopy with SMN2 RNA substrates, we discovered that the binding of two A1 molecules is crucial for splicing inhibition. One A1 may bind to the motif by C6T mutation in exon 7, while another may bind to both motifs via its two RRMs in intron 7. This inhibits U1 snRNP binding and affects the formation of complexes A and B. We propose a looping-out model to explain how A1 inhibits SMN2 exon 7 splicing. Additionally, we found that when SMN2 RNAs have a consensus 5ΚΉ splice site, the caused high levels of U1 snRNP binding overcome A1’s repressive effect on SMN2 RNA splicing and restore it by promoting the formation of complexes A and B.

History

Supervisor(s)

Ian Eperon; Andrew Hudson

Date of award

2023-07-18

Author affiliation

Department of Molecular and Cell Biology

Awarding institution

University of Leicester

Qualification level

  • Doctoral

Qualification name

  • PhD

Language

en