# Revealing molecular determinants of transcript-specific regulation in pre-mRNA splicing via rapid in vivo kinetic rate measurements

> **NIH NIH R01** · CORNELL UNIVERSITY · 2022 · $328,000

## Abstract

ABSTRACT
 It has long been known that pre-messenger RNA (pre-mRNA) splicing is an essential component of gene
expression in eukaryotic organisms, yet the past decade has seen a dramatic increase in our appreciation for its
role in regulating gene expression1. Most higher eukaryotes, including humans, regulate alternative splicing as
a tool for proteome expansion, and an ever-increasing number of human diseases are associated with mutations
in this pathway2,3. The mechanisms by which the spliceosome, which catalyzes pre-mRNA splicing, enacts this
regulation is a complex problem whose solution remains poorly understood yet will be critical to understanding
the etiology of many diseases. Proper regulation requires the spliceosome to faithfully assemble upon and
activate ‘cognate’ splice site sequences in the background of scores of aberrant, ‘near-cognate’ splice sites, yet
the spliceosome must balance this high fidelity splice site selection with the need for rapid, efficient splicing. At
the simplest level, improved knowledge of how the spliceosome achieves this balance will require understanding
both: (1) the landscape of cis-regulatory elements at splice sites that enable them to be distinguished as either
‘cognate’ or ‘non-cognate’; and (2) the mechanisms by which the spliceosome discriminates between such sites.
 In the work described here, we seek to better understand basic mechanisms of pre-mRNA splicing regulation
by leveraging a powerful methodology recently developed in my lab called Multiplexed Primer Extension
sequencing, or MPE-seq. Our approach is unique in that it allows for the genome-wide detection of pre-mRNA
splicing intermediates. By combining this technique with rapid metabolic RNA labeling techniques developed by
others, my group has now determined the in vivo rates of both chemical steps of pre-mRNA splicing across the
complement of spliced transcripts in budding yeast. Remarkably, these data reveal a wide variation among the
rates, both between the two steps for individual transcripts and between different transcripts. The goals of the
work described here are to leverage the information derived from these experiments to push our understanding
of the principles that underlie this regulation.

## Key facts

- **NIH application ID:** 10383702
- **Project number:** 5R01GM140082-02
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** JEFFREY A PLEISS
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $328,000
- **Award type:** 5
- **Project period:** 2021-04-05 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10383702

## Citation

> US National Institutes of Health, RePORTER application 10383702, Revealing molecular determinants of transcript-specific regulation in pre-mRNA splicing via rapid in vivo kinetic rate measurements (5R01GM140082-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10383702. Licensed CC0.

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