# Regulation of mRNA Processing

> **NIH NIH R01** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2020 · $502,852

## Abstract

Project Summary
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.

## Key facts

- **NIH application ID:** 9926720
- **Project number:** 5R01GM034277-35
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Phillip A Sharp
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $502,852
- **Award type:** 5
- **Project period:** 1984-12-01 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9926720

## Citation

> US National Institutes of Health, RePORTER application 9926720, Regulation of mRNA Processing (5R01GM034277-35). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9926720. Licensed CC0.

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