We and others have found that dynamic regulation of the kinetics of intron removal can function as a rate- limiting step in gene expression. This novel class of Detained Introns (DIs) are individual introns present in otherwise fully-spliced, polyadenylated transcripts that are held in the nucleus, likely resulting from the presence of a stalled spliceosome. Modulation of splicing components affects the accumulation of DIs and thus the level of mRNAs in the contexts of differentiation, growth, drug treatment, and mutations underlying cancer. To study this regulation, we propose to:
Aim 1 : Investigate the mechanisms controlling DIs and their contribution to gene expression.
Aim 1. 1: Biochemically purify and characterize spliceosome assembly on detained introns.
Aim 1. 2: Quantify the effect of intron detention on levels of productive mRNA in vivo. We have found recently that loss of CDK12 activity, which phosphorylates the carboxyl-terminal domain of RNA polymerase II on serine 2, enhances premature polyadenylation at intronic sites, resulting in a decrease in mature mRNA for a subset of genes. Drugs relatively specific for CDK12 are being tested in clinical trials. We propose to:
Aim 2 : Elucidate the mechanism of CDK12 mediated suppression of early intronic polyadenylation.
Aim 2. 1 Decipher the precise step(s) in the transcription cycle regulated by CDK12.
Aim 2. 2. Quantitatively assess the role of CDK12 in transcription elongation. We have found recently, utilizing an RNA-exosome conditional depletion system, that U1 snRNP suppresses premature polyadenylation of sense RNA in the region of the first stable nucleosome. This is the case for at least a third of all genes expressed in mouse embryonic stem cells. These genes are enriched for long CpG Islands with associated unstable nucleosomes and flanking stable nucleosomes. We proposed that this is a checkpoint for the highly processive polymerase complex and propose to:
Aim 3 : Determine the roles of stable nucleosome-associated premature RNA polyadenylation in transcription checkpoint and splicing regulation.
Aim 3. 1: Characterize the relationship between stable nucleosome-associated premature RNA polyadenylation, polymerase II pausing, and splicing signals.
Aim 3. 2: Investigate the differential mechanisms of TSS-proximal and nucleosome-associated polymerase II pausing and their contributions to premature polyadenylation, splicing, and transcription elongation.
Aim 3. 3: Determine the involvement of U1 snRNP and Myc oncogene in the nucleosome-associated transcription checkpoint.
The proposed research will advance the understanding of gene structure and expression, allowing improved diagnosis, prevention, and treatment of human diseases and thus improvement in public health. The specific focus is identifying steps in RNA splicing and the relationship between the transcription process and RNA processing. This science will be the basis of the discovery of new types of therapeutics and early intervention in diseases.
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