# Structural and functional studies of mRNA processing, stability and quality control

> **NIH NIH R35** · COLUMBIA UNIV NEW YORK MORNINGSIDE · 2024 · $792,768

## Abstract

Project Summary
Most eukaryotic messenger RNA precursors (pre-mRNAs) must undergo extensive co-
transcriptional processing in the nucleus before they can be exported to the cytoplasm
and function as mRNAs. The processing events include 5¢-end capping, splicing, and 3¢-
end cleavage and polyadenylation. The 3’-end processing of most pre-mRNAs requires
a large number of protein factors for its execution, including cleavage and
polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage
factors I and II, and poly(A) polymerase (PAP). The 3’-end processing machinery in
yeast has similarity to that in mammals, although there are also significant differences.
Replication-dependent histone pre-mRNAs contain a conserved stem-loop near their 3¢-
end and employ a distinct machinery for its processing, although it shares some key
protein factors with the canonical pre-mRNA 3¢-end processing machinery.
mRNA 5¢-end capping occurs early during transcription by RNA polymerase II, and it
was generally believed that capping always proceeds to completion. We discovered
earlier that the DXO/Rai1 family of proteins are part of a mRNA capping quality
surveillance mechanism. They can possess RNA 5¢-end pyrophosphohydrolase (PPH)
and decapping activities, and help remove incompletely capped mRNAs from cells.
Despite the extensive studies on these mRNA processing and quality control factors,
significant gaps remain in our knowledge on their molecular mechanisms of action.
During the previous funding period, we determined the structure by cryo-EM of an active,
fully-reconstituted human histone pre-mRNA 3¢-end processing machinery, the first
structure of an active processing machinery. We have also shown that the DXO/Rai1
and Nudix family of enzymes can remove nucleotide metabolite caps on RNAs, such as
NAD (deNADding), FAD (deFADding) and dephospho-CoA (deCoAping). These
successes provide an excellent foundation for the proposed project.
Our main goals for the current funding period are to produce new structural information
on pre-mRNA and snRNA 3¢-end processing machineries, and to understand the
molecular basis for the diverse decapping activities of the DXO/Rai1 and Nudix
enzymes. We will carry out structural studies on the protein factors and their complexes
by both cryo-EM and crystallography, and assess the structural observations by careful
biochemical and functional experiments. The proposed project will greatly enhance our
understanding of these important events in the mRNA lifecycle.

## Key facts

- **NIH application ID:** 10833030
- **Project number:** 5R35GM118093-09
- **Recipient organization:** COLUMBIA UNIV NEW YORK MORNINGSIDE
- **Principal Investigator:** LIANG TONG
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $792,768
- **Award type:** 5
- **Project period:** 2016-05-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10833030

## Citation

> US National Institutes of Health, RePORTER application 10833030, Structural and functional studies of mRNA processing, stability and quality control (5R35GM118093-09). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10833030. Licensed CC0.

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