# Molecular Mechanisms of Co-Transcriptional Ribonucleoprotein Assembly

> **NIH NIH K99** · JOHNS HOPKINS UNIVERSITY · 2022 · $89,829

## Abstract

PROJECT SUMMARY
RNAs are integral components of molecular machines that carry out all essential processes in gene expression.
To expand the functional landscape of these molecular machines, RNAs synergize with proteins to form large
complexes called ribonucleoproteins (RNPs). RNPs are formed initially during transcription, where synthesis of
the RNA is coupled to RNA folding and association of proteins. A possible consequence of this coupling is that
improper co-transcriptional folding may delay protein association, thereby hindering RNP assembly. Yet, RNPs
like the ribosome form within minutes in the cell suggesting that there are mechanisms to prevent misfolding or
slow assembly. The ribosome represents an ideal model system for studying co-transcriptional RNP assembly,
because it contains a highly structured RNA that must be properly folded and assembled to function. Decades
of studies on bacterial ribosome assembly have supported a model for assembly in which ribosomal protein
association is strictly hierarchical; however, recent evidence from my work and others suggests that while stable
incorporation may be hierarchical, underlying transient protein binding nonetheless influences the RNA folding
path. The mechanism for how proteins chaperone the RNA during transcription to accelerate assembly is
currently unclear. Furthermore, while a similar ordered assembly mechanism has been proposed for eukaryotic
ribosome assembly, it is likely that underlying protein binding dynamics also plays a role in guiding folding of the
RNA during transcription. This proposal aims to understand the molecular consequences that arise from coupling
between transcription, RNA folding, and ribosome assembly by measuring RNA folding directly during co-
transcriptional assembly (Aim 1) and visualizing protein association on nascent eukaryotic RNAs (Aim 2). To
examine RNA folding during transcription-coupled ribosome assembly in Aim 1, I will probe the RNA structure in
real time in vitro during transcription using dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-
MaPseq). This method will provide a complete picture of the folding pathway while the RNA is being synthesized
in the presence and absence of proteins, thereby allowing for dissection of the individual contributions to the
assembly mechanism. Studying RNA folding directly will be complemented by single-molecule experiments in
Aim 2 that directly examine protein/RNP binding kinetics. Specifically, I will examine binding of UtpA and U3
snoRNP in real time to nascent yeast ribosomal RNA. Transitioning to studying transcription-coupled ribosome
biogenesis in eukaryotes will provide new insight into how transient binding may be a common theme in RNP
assembly. Results from the K99 phase will be expanded upon in the independent phase to examine folding and
assembly of larger yeast assembly intermediates, such as the 5’ external transcribed spacer particle. In total,
these aims will advance our underst...

## Key facts

- **NIH application ID:** 10331029
- **Project number:** 5K99GM140204-02
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Margaret Louise Rodgers
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $89,829
- **Award type:** 5
- **Project period:** 2021-02-01 → 2022-08-01

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10331029

## Citation

> US National Institutes of Health, RePORTER application 10331029, Molecular Mechanisms of Co-Transcriptional Ribonucleoprotein Assembly (5K99GM140204-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10331029. Licensed CC0.

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