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Elucidation of structure-function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein

Phan, Chau Hong Duc
http://orcid.org/0000-0003-0666-0133
http://rave.ohiolink.edu/etdc/view?acc_num=osu1658404296117553

Abstract Details

2022, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Ribonuclease P (RNase P) catalyzes cleavage of the 5'-leader in precursor-transfer RNAs (pre-tRNAs). The ribonucleoprotein (RNP) form of RNase P includes one catalytic RNA and one to ten protein subunits depending on the domain of life. An unsolved question in the field pertains to the possible gains afforded by the increased protein complexity of the archaeal/eukaryotic variants given the notion that RNase P is a housekeeping enzyme with a primary conserved function in all life. Here, we employ two systems: (i) an in vitro reconstituted archaeal RNase P, whose protein subunits (POP5, RPP30, RPP21, RPP29, L7Ae) share eukaryotic homologs, and (ii) a native eukaryotic RNase P partially purified from mouse brains, to study how RNA and multiple protein subunits assemble into an RNP complex and how protein subunits affect the function and substrate specificity of RNase P. With the recent advances in cryo-electron microscopy, the structure of RNase P from Methanocaldococcus jannaschii (Mja), an archaeon, has been reported at 4.6-Å resolution. This structure showed for the first time that an archaeal RNase P exists as a dimer, where each monomer includes one RNA (RNase P RNA, RPR) and one copy each of the five protein subunits (RNase P Proteins, RPPs). Since this observation contradicts the higher-order structure that was reported before for a different type of archaeal RNase P, we have combined native mass spectrometry (MS), mass photometry, and biochemical assays to validate the dimeric formation of Mja RNase P holoenzyme in vitro. Using native MS, we established that one or two copies of L7Ae can bind to a double kink-turn in each RPR in the Mja RNase P holoenzyme. However, only one copy of L7Ae is required for optimal cleavage activity of the holoenzyme. Unexpectedly, we discovered that the protein-protein interactions between L7Ae and RPP21 could bypass the necessity for interactions between L7Ae and the RPR. By performing pre-tRNA cleavage assays with different Mja RPR kink-turn mutants (i.e., defective in L7Ae binding), we also inferred that L7Ae may exert its role on Mja RNase P cleavage activity through protein-protein interactions. Our findings highlight the importance of combining native MS, mass photometry, and biochemical assays to interpret and extend medium- to low-resolution cryo-EM structures. Given the importance of protein-protein interactions in RNP complexes, as exemplified by this work, computational approaches to predict the proteins in an RNP complex should consider RNA recognition determinants as well as the protein interactome. Human RNase P is responsible for the 3'-processing of two long non-coding (lnc) RNAs, MALAT1 and NEAT1, that are highly expressed in certain types of cancer. Using partially purified RNase P from mouse brains, whose RNA and protein subunits are related to those in human RNase P, we gained evidence that the specificity of the enzyme towards the canonical (pre-tRNAs) and non-canonical substrates (lncRNAs) might depend on the composition of the protein subunits of RNase P. This finding paves the way for future studies that seeks to identify the proteins responsible for modulating RNase P cleavage activity towards non-canonical substrates.
Venkat Gopalan (Advisor)
Vicki Wysocki (Committee Member)
Marcos Sotomayor (Committee Member)
Amanda Simcox (Committee Member)
197 p.
Biochemistry
Mja, RNase P, RNP

Recommended Citations

Citations

  • Phan, C. H. D. (2022). Elucidation of structure-function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1658404296117553

    APA Style (7th edition)

  • Phan, Chau Hong Duc. Elucidation of structure-function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein. 2022. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1658404296117553.

    MLA Style (8th edition)

  • Phan, Chau Hong Duc. "Elucidation of structure-function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein." Doctoral dissertation, Ohio State University, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=osu1658404296117553

    Chicago Manual of Style (17th edition)

osu1658404296117553
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