# Analysis of transcription and splicing coordination during erythropoeisis using single molecule RNA-seq

> **NIH NIH R21** · YALE UNIVERSITY · 2020 · $250,311

## Abstract

ABSTRACT/PROJECT SUMMARY
Although often studied as distinct entities, transcription and RNA processing are intricately linked in
eukaryotes. Our previous results in budding and fission yeasts show that the spliceosome can quickly and
efficiently remove introns as soon as Pol II synthesizes them. Nevertheless, sometimes splicing is completely
suppressed, rendering transcripts “dead end”. These unspliced nascent transcripts fail to undergo polyA
cleavage and are degraded. We hypothesize that the balance between “productive” (spliced and
polyadenylated) and “dead-end” transcripts determines gene expression in mammalian cells. Furthermore, this
mechanism is likely crucial under conditions of stress, because transcriptional readthrough is a frequent feature
of cellular stresses induced by infection, cancer, osmotic and oxidative stress, and other conditions.
The application brings together the complementary expertise of two investigators who are responding to an
RFA from NHLBI on normal biological mechanisms that provide cells with resilience. Dr. Neugebauer is a
biochemist with expertise in transcription and splicing, while Dr. Pillai is a hematopoietic biologist with
expertise in generation of erythroid populations and their characterization. Our proposal investigates the
coupling between transcription and RNA processing during production of red blood cells (erythropoiesis or EP).
Mature enucleated red blood cells emerge from immature hematopoietic progenitors after undergoing a highly
regulated differentiation program guided by numerous exogenous signals. This differentiation is characterized
by dramatic changes in the transcriptome, resulting in a mature red cell that is essentially a hemoglobin factory.
b-globin, the most abundant transcript in mature erythroid cells, has served as a critical model for pioneering
studies in pre-mRNA splicing and mRNA stability. We hypothesize that positive and negative feedback between
splicing and transcription are important determinants of erythroid maturation, which must be resilient to
physiological conditions (e.g. pregnancy, high altitude) that cause tissue hypoxia. The resulting “Stress EP”
increases red cell production in order to deliver more oxygen to the tissues.
We therefore propose to investigate co-transcriptional splicing dynamics, using erythropoiesis as a model
system. We will implement two custom nascent RNA-Seq strategies developed in the Neugebauer lab: Single
Molecule Intron Tracking (SMIT) and long read sequencing of nascent RNA. In Aim 1, we will utilize an in vitro
culture model of human erythropoietic differentiation in which primary CD34+ cells are cultured with
erythropoietin and other trophic factors to generate erythroid cells and test the above hypotheses. Aim 2 will
explore how co-transcriptional RNA processing may contribute to transcriptomic changes during stress EP. This
study thereby pioneers experimental systems that will allow us to pinpoint gene regulatory mechanisms that
rel...

## Key facts

- **NIH application ID:** 10072570
- **Project number:** 1R21HL150642-01A1
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Karla M Neugebauer
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $250,311
- **Award type:** 1
- **Project period:** 2020-08-01 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10072570

## Citation

> US National Institutes of Health, RePORTER application 10072570, Analysis of transcription and splicing coordination during erythropoeisis using single molecule RNA-seq (1R21HL150642-01A1). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10072570. Licensed CC0.

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