Protection of Drosophila development through the regulation of alternative splicing in the germline
Lead Research Organisation:
University of Cambridge
Department Name: Genetics
Abstract
PhD project strategic theme: Understanding the rules of life
This project aims to understand the mechanisms regulating alternative splicing in the germline that allow Drosophila development to occur normally. The primary focus will be on understanding the mechanism through which piRNAs regulate splicing of the P-element DNA transposon in the Drosophila germline, preventing a series of developmental defects (called hybrid dysgenesis) resulting from P-element transposition, which include sterility and high levels of mutation. The presence of maternally inherited piRNAs in the germline leads to retention of a P-element intron containing a premature stop codon, preventing full translation of the encoded transposase and suppressing transposition of the element. A number of factors that are known to modulate changes in chromatin states in classical piRNA pathways are implicated in repressing splicing of this intron, through previous work showing splicing derepression upon knockdown of these factors. Additionally, this repression is associated with a global increase in H3K9 trimethylation across the element. In studying this example of alternative splicing regulation, the aim is to understand the mechanism of how chromatin is involved in regulating splicing and which factors are required. Potential experiments to understand this may include dCas9-mediated recruitment of chromatin-modifying proteins to the element to assess splicing effects, mass spectrometry of P-element-associating factors in the presence/absence of this regulation and assessing changes in RNA polymerase II activity through GRO-Seq and assays using polymerase mutants with altered speed. Previous work has linked chromatin changes with alternative splicing in the cases of other genes, so if the mechanisms at play here can be elucidated, further work could determine whether they apply to other examples of alternative splicing regulation. There is also the potential to expand this work in order to more broadly understand how alternative splicing is regulated in the Drosophila germline to prevent developmental defects, through the study of splicing regulation in endogenous developmental genes.
This project aims to understand the mechanisms regulating alternative splicing in the germline that allow Drosophila development to occur normally. The primary focus will be on understanding the mechanism through which piRNAs regulate splicing of the P-element DNA transposon in the Drosophila germline, preventing a series of developmental defects (called hybrid dysgenesis) resulting from P-element transposition, which include sterility and high levels of mutation. The presence of maternally inherited piRNAs in the germline leads to retention of a P-element intron containing a premature stop codon, preventing full translation of the encoded transposase and suppressing transposition of the element. A number of factors that are known to modulate changes in chromatin states in classical piRNA pathways are implicated in repressing splicing of this intron, through previous work showing splicing derepression upon knockdown of these factors. Additionally, this repression is associated with a global increase in H3K9 trimethylation across the element. In studying this example of alternative splicing regulation, the aim is to understand the mechanism of how chromatin is involved in regulating splicing and which factors are required. Potential experiments to understand this may include dCas9-mediated recruitment of chromatin-modifying proteins to the element to assess splicing effects, mass spectrometry of P-element-associating factors in the presence/absence of this regulation and assessing changes in RNA polymerase II activity through GRO-Seq and assays using polymerase mutants with altered speed. Previous work has linked chromatin changes with alternative splicing in the cases of other genes, so if the mechanisms at play here can be elucidated, further work could determine whether they apply to other examples of alternative splicing regulation. There is also the potential to expand this work in order to more broadly understand how alternative splicing is regulated in the Drosophila germline to prevent developmental defects, through the study of splicing regulation in endogenous developmental genes.
Organisations
People |
ORCID iD |
| Felipe Karam Teixeira (Primary Supervisor) | |
| Eleanor Blake (Student) |