# Pre-mRNA splicing regulation is critical for controlling macrophage activation

> **NIH NIH R35** · TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR · 2021 · $370,738

## Abstract

PROJECT SUMMARY
 Despite the substantial impact pre-mRNA splicing has on gene expression outcomes, little is known
about how the spliceosome itself is modified and regulated during cellular reprogramming. Innate immune cells
like macrophages reprogram gene expression when they sense a “danger signal,” such as a pathogen,
organelle damage, or chemical signal, to combat the detected threat. While changes that occur
transcriptionally during macrophage activation are well characterized, almost nothing is known about how pre-
mRNA splicing is regulated following immune stimuli. The long-term goal of this project is to uncover how
macrophage activation modifies the spliceosome and to connect these changes with innate immune gene
expression outcomes. The spliceosome is a complex and dynamic macromolecular machine. Its ability to
recognize introns and catalyze their removal relies on numerous RNA binding proteins that recognize specific
sequences in exons and introns to “read” the splicing code. The central hypothesis of this proposal is that
during macrophage activation, post-translational modification of splicing factors directs assembly of a
specialized spliceosome characterized by a distinct cohort of protein-protein interactions that promotes the
innate immune gene expression program. In support of this model, phosphoproteomic experiments reveal that
30+ splicing factors, many with known regulatory roles, are phosphorylated or dephosphorylated at specific
serine residues following lipopolysaccharide (LPS)-dependent activation of macrophages. Experiments
interrogating one such factor, hnRNP M, show that LPS treatment triggers dephosphorylation concomitant with
its redistribution in the nucleus. Loss of hnRNP M by shRNA-mediated knockdown in macrophages alters
alternative splicing of a number of pre-mRNAs and leads to hyper-induction of important innate immune
transcripts, including the potent inflammatory mediator IL-6 and the key viral restriction factor Mx1. This
proposal expands upon these observations, looking globally at changes to the spliceosome following
macrophage activation. It will combine high-throughput approaches, including affinity purification-mass
spectrometry, phosphoproteomics, RNA-seq, and RNA CLIP-seq with targeted genetic and biochemical
experiments to implicate specific splicing factors in driving innate immune gene expression changes. This
research program will fill key gaps in our knowledge of how splicing is regulated following macrophage
activation and further our understanding of how the spliceosome reads and interprets the splicing code not only
during innate immune activation but also during other cellular reprogramming, including differentiation, stress,
starvation, and carcinogenesis.

## Key facts

- **NIH application ID:** 10240558
- **Project number:** 5R35GM133720-03
- **Recipient organization:** TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
- **Principal Investigator:** Kristin Leigh Patrick
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $370,738
- **Award type:** 5
- **Project period:** 2019-09-01 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10240558

## Citation

> US National Institutes of Health, RePORTER application 10240558, Pre-mRNA splicing regulation is critical for controlling macrophage activation (5R35GM133720-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10240558. Licensed CC0.

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