# Cross-regulation between transcription and pre-mRNA splicing

> **NIH NIH R01** · YALE UNIVERSITY · 2024 · $461,561

## Abstract

Abstract/Project Summary
The ubiquitous need for transcription and pre-mRNA processing in eukaryotes requires an understanding of
the mechanisms by which they occur simultaneously and regulate each other. Our long-term goals are to
determine the mechanisms that govern co-transcriptional intron removal and how co-transcriptional splicing
contributes to gene expression regulation. With previous support from this grant, my lab has pioneered
strategies for purifying and sequencing nascent RNA – the transient intermediates in transcription and RNA
processing – to quantify pre-mRNA splicing relative to the position of elongating RNA Polymerase II (Pol II) on
the gene. Most introns are removed as soon as they emerge from Pol II, indicating that Pol II and the spliceosome
are timed to act together and physically close to one another. Knowing this, our overall objectives are to (i)
elucidate the co-transcriptional mechanism of “all-or-none” RNA processing, in which individual nascent
transcripts are either fully spliced and cleaved at the 3’ end (all) or fully unspliced and uncleaved (none). My lab
discovered this recently by sequencing full-length nascent RNAs, and it is a major co-transcriptional regulatory
mechanism for b-globin gene expression. (ii) Determine how the potential for splicing becomes limited as
transcription proceeds and the nascent transcript gets longer. And (iii) Determine the fate and possible function
of the unprocessed transcripts. The central hypothesis is that the potential for nascent RNA to bind positive- and
negative-acting RNA binding proteins and/or to undergo intermolecular base-pairing and compaction increases
as the nascent chain grows during transcription. We will test this hypothesis by pursuing three specific aims: 1)
develop a method to determine the local base-pairing behavior of nascent RNA, using DMS chemistry and
nascent RNA purification. The results of this aim will tell us the degree to which nascent RNA becomes
transiently structured during transcription and if local structures correlate with positive or negative effects on
splicing, 2) identify activator and/or repressor proteins that accumulate on the growing nascent chain during
transcription, using biochemical purification and in vivo labeling approaches. 3) determine the nuclear response
to unprocessed “none” transcripts. Are they delayed in processing? Or will they be degraded? For this aim, we
have established the A-count method, which sequences the entire transcript from 5’ end to 3’ end, including the
whole length of the polyA tail, which will be counted by nucleotide type and number. Our preliminary results
show that the nuclear polyA binding protein PABPN1 is regulated by phosphorylation during mitosis,
suggesting differential fates for nuclear retained mRNAs with introns. PABPN1 is a protein mutated in
Oculopharyngeal muscular dystrophy (OPMD). The proposed research is significant because these mechanisms
operating at the level of pre-mRNA processin...

## Key facts

- **NIH application ID:** 10910147
- **Project number:** 5R01GM112766-10
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Karla M Neugebauer
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $461,561
- **Award type:** 5
- **Project period:** 2015-09-01 → 2027-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10910147

## Citation

> US National Institutes of Health, RePORTER application 10910147, Cross-regulation between transcription and pre-mRNA splicing (5R01GM112766-10). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10910147. Licensed CC0.

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