RNA—Protein Structure-Function Relationships

We primarily use RNase P, a catalytic ribonucleprotein (RNP), as an experimental model to understand how proteins modulate RNA structure, thereby broadening the functional repertoire of RNP enzymes. We are especially interested in mechanistic insights that can be used to understand RNA—protein cooperation and how dysfunction in this regard can lead to disease.

I. NIH 1R01GM120582-01 (October 2016 – July 2020)

Title
Dissecting functional cooperation among subunits in a catalytic ribonucleoprotein

Narrative
A number of genetic diseases (e.g., neurodegeneration) are associated with defective processing of transfer RNAs, which are needed to decode the genetic blueprint. One of the enzymes critical for this processing (i.e., RNase P) depends on cooperation between a catalytic RNA and multiple protein subunits. To understand this enzyme’s assembly and catalysis, we will study how the protein subunits affect the structure and function of its essential RNA core, thus contributing to a mechanistic appreciation that could help elucidate the causes of disease.

Senior personnel
•   Venkat Gopalan (OSU, lead PI)
•   Mark P. Foster, Michael G. Poirier, Vicki H. Wysocki (OSU, co-Is)
•   Julius B. Lucks (Northwestern, co-I)

Lab members involved in this project
•   Lien B. Lai (Senior Research Scientist)
•   Stella M. Lai (Research Associate)
•   Edric K. Choi (Research Assistant)
•   James Li (Research Assistant)
•   Walter J. Zahurancik (Postdoctoral Researcher)
•   Ila A. Marathe (Graduate Student, OSU Microbiology Graduate Program)
•   Hong-Duc Phan (Graduate Student, Ohio State Biochemistry Program)
•   Vaishnavi Sidharthan (Graduate Student, Chemistry and Biochemistry Graduate Program)

Methods
•   In our laboratory: site-directed mutagenesis; RNA (in vitro) transcription, purification, and labeling; protein overexpression and purification; RNA processing assays; enzyme kinetic studies; hydroxyl radical-mediated RNA—protein footprinting; ensemble fluorescence (FRET) assays; fluorescence polarization (FP) binding assays
•   In collaboration with other PIs: electron paramagnetic resonance (EPR) spectroscopy (Foster); selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE)-seq (Lucks); single-molecule fluorescence microscopy (Poirier); mass spectrometry (Wysocki)

Recent Publications
*Corresponding authors
•   Lai SM and Gopalan V*. (2019) Using an L7Ae-tethered, hydroxyl radical-mediated footprinting strategy to identify and validate kink-turns in RNAs. Methods Mol. Biol., in press.
•   Palsule G, Gopalan V, and Simcox A*. (2019) Biogenesis of RNase P RNA from an intron requires co-assembly with cognate protein subunits. Nucleic Acids Res., 47: 8746-8754. [SI]
•   Daniels CJ*, Lai LB, Chen T-H, and Gopalan V. (2019) Both kinds of RNase P in all domains of life: Surprises galore. RNA, 25: 286-291.
•   Gopalan V*, Jarrous N*, and Krasilnikov AS*. (2018) Chance and necessity in the evolution of RNase P. RNA, 24: 1-5.
•   Lai LB*, Tanimoto A^, Lai SM^, Chen W-Y, Marathe IA, Westhof E, Wysocki VH, and Gopalan V*. (2017) A novel double kink-turn module in euryarchaeal RNase P RNAs. Nucleic Acids Res., 45: 7432-7440. [SI] ^joint second authors
•   Samanta MP°*, Lai SM°, Daniels CJ, and Gopalan V*. (2016) Sequence analysis and comparative study of the protein subunits of archaeal RNase P. Biomolecules, 6: 22. [SI] °joint first authors
•   Lai SM, Lai LB, Foster MP*, and Gopalan V*. (2014) The L7Ae protein binds to two kink-turns in the Pyrococcus furiosus RNase P RNA. Nucleic Acids Res., 42: 13328-13338. [SI]
•   Ma X°, Lai LB°, Lai SM°, Tanimoto A, Foster MP, Wysocki VH*, and Gopalan V*. (2014) Uncovering the stoichiometry of Pyrococcus furiosus RNase P, a multi-subunit catalytic ribonucleoprotein complex, by surface-induced dissociation and ion mobility mass spectrometry. Angew. Chem. Int. Ed. Engl., 53: 11483-11487. [SI] °joint first authors

II. United States-Israel Binational Science Foundation #2015/157 (October 2016 – September 2020)

Title
Role of human RNase P in transcription and processing of tRNA

Senior personnel
•   Nayef Jarrous (The Hebrew University of Jerusalem, lead PI)
•   Sidney Altman (Yale University, co-PI)
•   Venkat Gopalan (OSU, co-PI)

Lab members involved in this project
•   Edric K. Choi (Research Assistant)

Methods
•   In our laboratory: RNA (in vitro) transcription, purification, and labeling; protein overexpression and purification; chromatin immunoprecipitation sequencing (ChIP-Seq)

Recent Publications
•   Gopalan V*, Jarrous N*, and Krasilnikov AS*. (2018) Chance and necessity in the evolution of RNase P. RNA, 24: 1-5.

III. Ohio Agricultural Research and Development Center #OHOA1627 (September 2018 – August 2020)

Title
Genome-wide mapping of plant tandem zinc-finger protein—RNA interactions for crop improvement

Senior personnel
•   Jyan-Chyun Jang (OSU, lead PI)
•   Venkat Gopalan (OSU, co-PI)