Projects and Grants
Post-transcriptional Regulation of Gene Expression
The mechanism of RNA processing, and particularly messenger RNA decay, in the Gram-positive bacterium Bacillus subtilis is the major focus of our laboratory. We have shown that the 5’ end of a message plays an important role in determining mRNA half-life, and are currently looking at specific sites on the mRNA at which decay initiates. This is being pursued from the point of view of the ribonucleases involved in mRNA decay, and from the point of view of the mRNA itself (sequence, structure).
Several genes encoding ribonucleases have been cloned, including four genes encoding 3’-to-5’ exonucleases and one gene encoding a narrow-specificity endonuclease (called “Bs-RNase III”). In order to understand the function of these exonucleases, we are analyzing decay of endogenous mRNAs in mutant strains lacking one or more of these genes. We are concentrating on small, monocistronic mRNAs, as these are amenable to detailed analysis and likely provide few sites for initiation of decay.
Recent progress in the study of mRNA decay in B. subtilis has led to the discovery of a new class of ribonucleases, called RNase J (Fig. 1). Amazingly, enzymes in this class have both endonucleolytic and 5’ exonucleolytic activities. B. subtilis has two RNase J enzymes, RNase J1 and J2, and RNase J1 is essential. We have found that the 5’ exonuclease activity of RNase J1 is sensitive to the phosphorylation state of the 5’ end; RNase J1 degrades an mRNA with a 5’-monophosphate end but not an mRNA with a 5’-triphosphate end. Based on the characteristics of RNase J1, a model for mRNA turnover in B. subtilis, which differs from the accepted model in E. coli, has been developed (Fig. 2). Ongoing in vitro and in vivo studies are aimed at understanding the role of RNase J1 in global mRNA decay. The 140-nucleotide trp leader RNA has proved to be a valuable model, and our studies have allowed us to describe in complete detail the turnover of this RNA (Fig. 3). In 2009, another essential endonuclease has been discovered in B. subtilis, named RNase Y. We have recently published the first report of a specific messenger RNA whose stability is determined by RNase Y.
Collaborations with other laboratories are in progress on the following projects:
- The effect of polyadenylation on mRNA decay. We have demonstrated that B. subtilis mRNA is polyadenylated, but the identity of the B. subtilis poly(A) polymerase has been a mystery. Biochemical purification of a poly(A) polymerase activity is anticipated, which will be followed by genetic studies of a strain lacking this activity.
- Removal of the inhibiting 5’-triphosphate end. It has been demonstrated in E. coli that removal of a pyrophosphate group to give a monophosphate 5’ end can be an important step in the initiation of mRNA decay. Based on genome homology predictions, B. subtilis contains at least six putative pyrophosphohydrolase genes, and these are being evaluated for their role in mRNA decay.
- In vitro activity of RNase J1. Numerous RNAs synthesized in vitro are being used to examine the properties of RNase J1. With the appropriate conditions, these test RNAs can be labeled at their 5’ end, their 3’ end, or internally, and analysis of the products of digestion of these RNAs with RNase J1 will be helpful in revealing how RNase J1 binds to and cleaves its target RNAs.
David Bechhofer, PhD
19th Floor, Room 19-98
Pharmacology and Systems Therapeutics
1468 Madison Avenue
New York NY, 10029