Tuesday, July 1, 2025 7/1/2025

Stimulation of Ribosomal Frameshifting by Cotranslational Membrane Protein Folding and Misfolding

Award Number: R01GM138845
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 02/01/2021
PERIOD OF PERFORMANCE END DATE: 12/31/2024

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Stimulation of Ribosomal Frameshifting by Cotranslational Membrane Protein Folding and Misfolding - ABSTRACT The proteostasis network relies on numerous feedback mechanisms to strike a balance between the rates of protein synthesis and degradation, which is crucial for the maintenance of protein homeostasis. Proper tuning of the rate of protein synthesis is also critical for the fidelity of cotranslational protein folding, which requires coordination between the ribosome and various molecular chaperones. This translational regulation is especially important for the fidelity of membrane protein (MP) biosynthesis, as the disruption of translational dynamics appears to coincide with cotranslational misfolding and premature degradation. Nevertheless, it is currently unclear how the translational machinery detects and responds to the cotranslational MP misfolding. In a recent study of the topological properties of the Sindbis virus (SINV) structural polyprotein, our team found that the translocon-mediated membrane integration of the nascent polypeptide stimulates ribosomal frameshifting and the premature termination of translation. This work revealed that cotranslational (mis)folding can alter translation through programmed ribosomal frameshifting (PRF), which is typically viewed as an RNA-mediated translational recoding mechanism. In the following, we outline evidence suggesting translocon-mediated PRF occurs during the translation of many human MPs, including several misfolding-prone MPs such as the cystic fibrosis transmembrane conductance regulator (CFTR). We provide multiple lines of evidence that demonstrate that PRF can occur at several “checkpoints” during CFTR synthesis, and show that a pathogenic mutation known to induce cotranslational misfolding (ΔF508) stimulates ribosomal frameshifting and the premature termination of CFTR translation. Based on these findings, we hypothesize that PRF sites allow the ribosome to tune the processivity of translation in response to conformational transitions in the nascent chain. To test this hypothesis, we will assess how mutations and small molecules that alter cotranslational CFTR folding impacts the processivity of translation at each PRF site. To gain structural insights into this ribosomal frameshifting mechanism, we will also extend our studies on the SINV structural polyprotein. To map the sequence constraints of translocon-mediated PRF, we measured the effects of 2,003 mutations on the efficiency of ribosomal frameshifting by deep mutational scanning. Our preliminary results reveal several structural features that appear to be critical for PRF, including a putative lipid-binding face within a nascent transmembrane domain and a helical segment within the ribosomal exit tunnel. To determine how these structural features induce PRF, we propose a novel fusion of molecular modeling, cellular biochemistry, and virology experiments to elucidate these structural features. Finally, we will leverage these insights to develop sequence-based energetic predictions for the efficiency of PRF within integral MPs. We will also characterize putative PRF sites in several disease-linked MPs in order to validate these findings and explore the potential role of PRF in MP homeostasis. Together, these investigations will provide fundamental insights into a novel cotranslational feedback mechanism and the molecular basis of disease.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = -$86 )
2025	2024	PURDUE UNIVERSITY	2550 NORTHWESTERN AVE # 1100	WEST LAFAYETTE	IN	47906	TIPPECANOE	USA	Biomedical Research and Research Training	001	4	4/14/2025	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	-$86
2025	2023	TRUSTEES OF INDIANA UNIVERSITY	107 S INDIANA AVE	BLOOMINGTON	IN	47405	MONROE	USA	Biomedical Research and Research Training	000	3	1/19/2025	NON-COMPETING CONTINUATION	$0
														Subtotal = -$86

Issue Date FY: 2024 ( Subtotal = $318,073 )
2024	2024	PURDUE UNIVERSITY	2550 NORTHWESTERN AVE # 1100	WEST LAFAYETTE	IN	47906	TIPPECANOE	USA	Biomedical Research and Research Training	002	4	7/24/2024	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$31,807
2024	2024	PURDUE UNIVERSITY	2550 NORTHWESTERN AVE # 1100	WEST LAFAYETTE	IN	47906	TIPPECANOE	USA	Biomedical Research and Research Training	001	4	1/31/2024	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$286,266
2024	2023	TRUSTEES OF INDIANA UNIVERSITY	107 S INDIANA AVE	BLOOMINGTON	IN	47405	MONROE	USA	Biomedical Research and Research Training	000	3	1/30/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $318,073

Issue Date FY: 2023 ( Subtotal = $304,820 )
2023	2023	TRUSTEES OF INDIANA UNIVERSITY	107 S INDIANA AVE	BLOOMINGTON	IN	47405	MONROE	USA	Biomedical Research and Research Training	000	3	12/9/2022	NON-COMPETING CONTINUATION	$274,338
2023	2023	TRUSTEES OF INDIANA UNIVERSITY	107 S INDIANA AVE	BLOOMINGTON	IN	47405	MONROE	USA	Biomedical Research and Research Training	001	3	2/15/2023	NON-COMPETING CONTINUATION	$30,482
														Subtotal = $304,820

Issue Date FY: 2022 ( Subtotal = $305,061 )
2022	2022	TRUSTEES OF INDIANA UNIVERSITY	107 S INDIANA AVE	BLOOMINGTON	IN	47405	MONROE	USA	Biomedical Research and Research Training	001	2	6/29/2022	NON-COMPETING CONTINUATION	$30,507
2022	2022	TRUSTEES OF INDIANA UNIVERSITY	107 S INDIANA AVE	BLOOMINGTON	IN	47405	MONROE	USA	Biomedical Research and Research Training	000	2	12/8/2021	NON-COMPETING CONTINUATION	$274,554
														Subtotal = $305,061

Issue Date FY: 2021 ( Subtotal = $319,921 )
2021	2021	TRUSTEES OF INDIANA UNIVERSITY	107 S INDIANA AVENUE	BLOOMINGTON	IN	47405	MONROE	USA	Biomedical Research and Research Training	000	1	1/26/2021	NEW	$319,921
														Subtotal = $319,921

Grand Total All Awards = $1,247,789

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Stimulation of Ribosomal Frameshifting by Cotranslational Membrane Protein Folding and Misfolding

Award Number: R01GM138845

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 02/01/2021

PERIOD OF PERFORMANCE END DATE: 12/31/2024

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