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Eunice Kennedy Shriver National Institute of Child Health and Human Development - Maraia Lab - Section on Molecular and Cell Biology
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Eukaryotic tRNA Expression

It has been estimated that "the average tRNA molecule interacts with probably 30-40 proteins during its life cycle." (Söll, D. "Transfer RNA: An RNA for All Seasons" in The RNA World, Gesteland R.F. and Atkins, J.F. [eds] CSHL Press, 1993). Newly synthesized precursor tRNAs must undergo 5' end-processing, intron removal, base modification, and 3' end processing, 3' CCA addition, acylation, and nuclear export, before it is functional. Understanding the mechanisms by which La and pol III-associated factors function in the pathways used for small RNA production, and how these pathways are controlled during normal cellular proliferation are major goals of this Section. We have therefore been working to establish a genetically tractable system to study this in the fission yeast, Schizosaccharomyces pombe.


Development of a genetic system to study pol III transcription, including termination and La function, in fission yeast. The budding yeast, S. cerevisiae and human share extensive conservation in the factors and mechanisms involved in transcription initiation but exhibit significant differences in the TFs that interact with the downstream promoter as well as the sequence element used for termination. Therefore, another goal of this Section is to extend our investigations of pol III transcription to the fission yeast, S. pombe. Preliminary data suggest a pol III termination signal recognition mechanism for S. pombe that is more similar to that of humans than is S. cerevisiae.

A pol III-dependent reporter gene was developed and characterized for use in S. pombe. This gene encodes an opal suppressor tRNA that can suppress a nonsense codon in the mRNA encoding a purine-synthetic enzyme (ade6-704) whose activity can be monitored by an in vivo colorimetric plate assay. The expression of this gene in S. pombe is dependent on accurate and efficient termination by pol III (see figure). This tRNA gene requires La for efficient expression in vivo (not shown). This system is allowing us to isolate factors involved in tRNA expression. That tRNA expression may be regulated by mechanisms that operate at the transcriptional post-initiation level in eukaryotes (as suggested by our studies on a human tRNA gene and human La) and that we may be able to identify the regulatory factors involved in this process are exciting possibilities that we are currently exploring. Perhaps more importantly however, this system should be able to provide the means to identify and manipulate the intracellular signaling pathways that regulate the tRNA expression pathway. This is important because it had not been appreciated that tRNA maturation was a regulated process in eukaryotes. The significance of this, if true, is that we may be able to learn more about how the cell regulates the most fundamental of the factors used to carry out the genetic program, tRNAs. Without tRNAs there can be little chance for cellular proliferation. Therefore, how these mechanisms may be used to control proliferation in cancer cells will have to be explored. This is a promising area for exploration. We have also been developing an S. pombe-derived in vitro pol III transcription system to complement our studies in vivo. We are very enthusiastic about the promise this system represents.

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