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Peer-reviewedCC BY 4.0Nucleic Acids Research

Structural probing of RNA hairpins quantifies protein occupancy on RNA and links it to function in human cells

  • Abby R Thurm1
  • Tanya H Nguyen2
  • Jesus Damian Torres Campos2
  • Matthew Mansfield2
  • Lacramioara Bintu3
  • Lauren D Hagler2
  1. 1Biophysics Program, Stanford University School of Medicine , Stanford, CA 94305 ,
  2. 2Department of Chemistry, Texas A&M University, College Station , TX 77843 ,
  3. 3Department of Bioengineering, Stanford University , Stanford, CA 94305 ,
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Abstract

RNA structures that form inside living cells govern processes ranging from translation to RNA decay, many of which are controlled by RNA-binding proteins (RBPs). Because RBP specificity can depend on both RNA sequence and folding, traditional pulldown-based methods often obscure the structural context of bound RNAs. Here, a quantitative framework based on dimethyl sulfate mutational profiling and sequencing was established to directly measure RNA structure and RBP binding in human cells at single-nucleotide resolution. Applied to a 1600-member MS2 hairpin library, this approach revealed that RNA folding is the strongest determinant of MS2 coat protein (MCP) recognition in the MS2–MCP system, requiring formation of a stable hairpin and a consensus loop sequence, with stem length or GC content contributing primarily through their effect on hairpin folding. An in-cell occupancy model was derived to quantify fractional binding from structural readouts. Complementary systems using degron-tuned MCP and a downregulatory MCP fusion demonstrated binding behavior under subsaturating protein concentrations and showed that inferred occupancy quantitatively predicts RNA degradation efficiency. Together, these results establish a quantitative strategy for measuring RBP–RNA specificity and affinity directly in cells, using the MS2–MCP system to link RNA structure, protein occupancy, and regulatory outcome.

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Cite

BibTeXRIS
BibTeX
@article{thurm2026structural,
  title = {{Structural probing of RNA hairpins quantifies protein occupancy on RNA and links it to function in human cells}},
  author = {Abby R Thurm and Tanya H Nguyen and Jesus Damian Torres Campos and Matthew Mansfield and Lacramioara Bintu and Lauren D Hagler},
  journal = {Nucleic Acids Research},
  year = {2026},
  month = oct,
  doi = {10.1093/nar/gkag1019},
  url = {https://academic.oup.com/nar/article/doi/10.1093/nar/gkag1019/9005089},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Structural probing of RNA hairpins quantifies protein occupancy on RNA and links it to function in human cells
AU  - Abby R Thurm
AU  - Tanya H Nguyen
AU  - Jesus Damian Torres Campos
AU  - Matthew Mansfield
AU  - Lacramioara Bintu
AU  - Lauren D Hagler
PY  - 2026
DA  - 2026/10/01
JO  - Nucleic Acids Research
DO  - 10.1093/nar/gkag1019
UR  - https://academic.oup.com/nar/article/doi/10.1093/nar/gkag1019/9005089
AB  - RNA structures that form inside living cells govern processes ranging from translation to RNA decay, many of which are controlled by RNA-binding proteins (RBPs). Because RBP specificity can depend on both RNA sequence and folding, traditional pulldown-based methods often obscure the structural context of bound RNAs. Here, a quantitative framework based on dimethyl sulfate mutational profiling and sequencing was established to directly measure RNA structure and RBP binding in human cells at single-nucleotide resolution. Applied to a 1600-member MS2 hairpin library, this approach revealed that RNA folding is the strongest determinant of MS2 coat protein (MCP) recognition in the MS2–MCP system, requiring formation of a stable hairpin and a consensus loop sequence, with stem length or GC content contributing primarily through their effect on hairpin folding. An in-cell occupancy model was derived to quantify fractional binding from structural readouts. Complementary systems using degron-tuned MCP and a downregulatory MCP fusion demonstrated binding behavior under subsaturating protein concentrations and showed that inferred occupancy quantitatively predicts RNA degradation efficiency. Together, these results establish a quantitative strategy for measuring RBP–RNA specificity and affinity directly in cells, using the MS2–MCP system to link RNA structure, protein occupancy, and regulatory outcome.
SN  - 0305-1048
SN  - 1362-4962
ER  -