Ribonuclease P (RNase P) is a ubiquitous and essential ribonucleoprotein (RNP) involved in 5Õ maturation of transfer RNAs (tRNAs). Bacterial RNase P consists of a catalytic RNA subunit and a protein cofactor. While the RNA subunits of most archaeal and all eukaryal RNase P contain several of the critical and conserved secondary structural elements present in their bacterial counterpart, they are not catalytically active in vitro in the absence of their multiple protein subunits, whose number varies from four to ten depending on the source. Since both the RNA and protein subunits are essential for function in vivo in all three domains of life, RNase P serves as a paradigm for understanding how proteins modulate RNA function and for evaluating if RNase P represents an early RNA enzyme progressing through evolutionary stages to an RNA-protein enzyme in which proteins have usurped most of the functions originally carried out by the RNA moiety. There are three projects in the Gopalan lab that are encompassed within these long-term objectives.

1.  Bacterial RNase P

 

Bacterial RNase P consists of a catalytic RNA subunit (termed M1 RNA in Escherichia coli) that is catalytic and a lone protein cofactor (called C5 protein) that plays a supporting and essential role in precursor tRNA (ptRNA) processing in vivo. Kinetic studies have established that C5 protein facilitates substrate binding and enhances the rate of chemical cleavage by M1 RNA. Our overall goal is to elucidate the structural basis for these effects of C5 protein and use this simple RNP complex as a paradigm for understanding how proteins modulate RNA catalysis.

 

Project Participants: Lien Lai

 

Funding agencies: American Heart Association, National Science Foundation

2.  Archaeal RNase P

 

This project is a collaborative effort with Prof. Mark FosterÕs team at OSU. Although the multiple subunits of eukaryal RNase P have now been established, biochemical reconstitution of the ten or so subunits has proven difficult. Archaeal RNase P, albeit related to eukaryal RNase P, is a simpler RNP complex and has proven much more amenable to biochemical and structural investigations. Our goal is to use a multi-disciplinary approach (NMR spectroscopy, chemical and nuclease mapping, crosslinking, mass spectrometry, etc.) to elucidate the assembly and structure-function relationships in archaeal RNase P.

 

Project Participants: Wen-Yi Chen, I-Ming Cho, Lien Lai, Dileep Pulukkunat

 

Funding agencies: National Institutes of Health

3.  Plant RNase P

 

Since very little is known about the biochemical nature of plant RNase P, our first objective is to identify and characterize the RNA and protein subunits of Arabidopsis thaliana RNase P. A combination of database mining and novel cDNA isolation procedures has already proven partly fruitful in this regard. Complete identification of all the plant RNase P subunits will be accomplished by ongoing affinity purification and proteomics-based approaches. We expect T-DNA insertion mutant Arabidopsis lines will enable us to assess the altered phenotypes in plants that result from the loss of function of a specific subunit of plant RNase P.

 

The plant RNase P project also involves exploring the use of this enzyme for targeted degradation of endogenous RNAs. Specifically, the method involves the expression of a small RNA called an external guide sequence (EGS) which when bound to the target RNA entices RNase P to cleave the target RNA and thereby disrupt its expression. This method for specific inhibition of gene expression is currently being tested in Arabidopsis and maize. Preliminary results lend merit to optimization of this method as a viable functional genomics approach.

 

Project Participants: Cecilia Go, Lien Lai

 

Funding agencies: National Science Foundation

 

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