Tuesday, January 14, 2025
1/14/2025
PROJECT SUMMARY: Precise control of gene expression during development and in response to signals is essential for organismal growth and homeostasis. Accordingly, gene expression is regulated at multiple steps, with tight control over transcription elongation by RNA polymerase II (RNAPII) and coordinated processing of messenger RNA (mRNA). Recent work by our lab and others has revealed that elongating RNAPII can be targeted for premature termination. Intriguingly, across eukaryotes, mammals display an increased prevalence of premature termination and “transcription attrition” within mRNAs. This phenomenon is enriched among genes involved in signaling, the DNA damage response, development and tissue-specific functions. Although the reasons for this remain to be defined, genes in these classes are often long and harbor extensive first introns, leading to suggestions that intron expansion during evolution enabled the acquisition of cryptic termination-promoting sequences. Clearly, full-length mRNA synthesis is essential for proper protein production. Accordingly, intronic termination has emerged as a contributor to many diseases, including immune dysfunction, neurodegeneration and cancer. Here, we propose to systematically define the cis-acting sequences and trans-acting protein factors that determine the fate of the RNAPII elongation complex and nascent RNA. We will define how RNAPII elongation is regulated at mRNA loci to prevent inappropriate 3’ end formation and production of aberrant transcripts, and conversely, how transcription of enhancer and antisense RNAs is rapidly terminated to prevent generation of unwanted non-coding RNA (ncRNA) species. To accomplish these goals, we developed synergistic in vivo and in vitro systems. In Aim 1, we will use a powerful screening strategy in mouse embryonic stem cells to define the sequences and proteins that influence RNAPII elongation properties and RNA fate. To complement these cell-based approaches, Aim 2 will make use of a novel cell-free transcription system to dissect the biochemical mechanisms that control RNAPII elongation and the interplay with RNA processing and termination complexes. Aim 3 will build on our preliminary data demonstrating that transcription through the first exon-intron junction stimulates RNAPII elongation rate, to investigate in detail how the sequences and protein factors involved in splicing impact RNAPII activity. This work will answer central questions about the nature of termination-promoting sequences and the factors that govern their recognition, and will describe the interactions between elongation, splicing and termination complexes. These studies will identify the requirements for elongation of a functional mRNA and the mechanisms employed to prevent transcription attrition. In parallel, we will uncover the sequences and factors that promote early termination at enhancers and other regulatory loci to prevent polymerase collisions, double-stranded RNA formation and genome instability. By elucidating these mechanisms of RNAPII control we aim to reveal the causes of, and suggest potential treatments for, the growing list of diseases involving disruption of transcription elongation, splicing and 3’ end choice.
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