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

> **NIH NIH R35** · COLUMBIA UNIV NEW YORK MORNINGSIDE · 2022 · $90,293

## Abstract

Project Summary (from the funded MIRA application)
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.
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## Key facts

- **NIH application ID:** 10580942
- **Project number:** 3R35GM118093-07S1
- **Recipient organization:** COLUMBIA UNIV NEW YORK MORNINGSIDE
- **Principal Investigator:** LIANG TONG
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $90,293
- **Award type:** 3
- **Project period:** 2016-05-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10580942

## Citation

> US National Institutes of Health, RePORTER application 10580942, Structural and functional studies of mRNA processing, stability and quality control (3R35GM118093-07S1). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10580942. Licensed CC0.

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