(734g) New Tools for Controlling Gene Expression in Drosophila Melanogaster Embryos Using Hammerhead Ribozymes and Short Open Reading Frames

Authors: 
Jermusyk, A., North Carolina State University
Muller, P., North Carolina State University
Jacobsen, T., North Carolina State University
Beisel, C. L., North Carolina State University
Reeves, G. T., North Carolina State University

There are many situations where it is desirable to finely tune gene expression levels, including the study of both native and synthetic gene networks.  Two methods that are currently being explored are hammerhead ribozymes and short upstream open reading frames.  These two systems work through very different mechanisms.  The hammerhead ribozyme leads to mRNA degradation through autocatalytic self-cleavage, resulting in changes in both mRNA and protein levels (Yen et al. 2004, Auslander, Ketzer & Hartig 2010).  The sequences directly upstream of the ribozyme can be altered to adjust the ability of the ribozyme to be properly folded and thereby altering the amount of mRNA degradation (Yen et al. 2004, Auslander, Ketzer & Hartig 2010).  In the short upstream open reading frame approach, three upstream open reading frames (uORFs) encoding two amino acids were placed upstream of the open reading frame encoding the protein of interest.  Only a small number of ribosomes are able to “leak” past the uORFs to the ORF for the protein of interest (Ferreira, Overton & Wang 2013).  A different number of uORF and altering the surrounding sequences of these uORF can be used to alter the amount of ribosomes that “leak” through and translate the downstream ORF, producing the protein of interest (Ferreira, Overton & Wang 2013).  Both of these systems have been previously tested in single-cell systems to various extents (Auslander, Ketzer & Hartig 2010, Ferreira, Overton & Wang 2013).  We further validated these systems and showed their ability to regulate gene expression in a multi-cellular system, namely the Drosophila melanogaster embryo.  We were able to demonstrate the tuning of gene expression of EGFP within the embryo at both the protein and mRNA level for the hammerhead ribozyme and at the protein level for the uORFs.  Our results can be used to tune protein or mRNA level of endogenous or exogenous genes in Drosophila melanogaster or other multi-cellular system to a known level based on the insertion of a ribozyme or uORF.

References:

Auslander, S., Ketzer, P. & Hartig, J.S. 2010, "A ligand-dependent hammerhead ribozyme switch for controlling mammalian gene expression.", Mol Biosyst, vol. 6, no. 5, pp. 807-814.

Ferreira, J.P., Overton, K.W. & Wang, C.L. 2013, "Tuning gene expression with synthetic upstream open reading frames", Proceedings of the National Academy of Sciences of the United States of America, vol. 110, no. 28, pp. 11284-11289.

Yen, L., Svendsen, J., Lee, J., Gray, J.T., Magnier, M., Baba, T., D'Amato, R.J. & Mulligan, R.C. 2004, "Exogenous control of mammalian gene expression through modulation of RNA self-cleavage.", Nature, vol. 431, no. 7007, pp. 471-476.