Research Areas
1. Spliced leader RNA trans-splicing in metazoa
- Functional significance of trans-splicing
- Post-transcriptional role of SL addition in mRNA translation and stability
- Structure/function of mRNA cap-binding or interacting proteins (eIF4E, eIF4G, nuclear cap binding complex, decapping, etc.) and their role in mRNA metabolism
2. Development of molecular genetic tools for parasitic helminths (Molecular Parasitology)
1. Spliced leader RNA trans-splicing in metazoa
A major goal of our research is to understand the functional significance of spliced leader RNA trans-splicing. Spliced leader trans-splicing accurately joins sequences derived from separately transcribed RNAs, a spliced leader RNA (SL RNA) and a pre-mRNA. A spliced leader sequence (SL) is donated from a small RNA, the spliced leader RNA, to pre-mRNAs to form the 5' terminal exon of the mature mRNA (see Fig. A ). With addition of the mRNA 5' terminal SL sequence, trans-splicing also contributes a trimethylguanosine cap (m 2,2,7 GpppG, trimethylguanosine = TMG) to metazoan mRNAs.
A. Spliced Leader Trans-Splicing contributes a Unique Cap and Sequence to the 5' end of Recipient mRNAs
B. m 7 GpppG (MMG-cap) vs m 2,2,7 GpppG (TMG-cap)
The primary function of metazoan trans-splicing remains unknown.
Our working hypothesis is that both the SL sequence and cap structure likely play an important role in the metabolism
(e.g., pre-mRNA processing, mRNA export, translation, and stability) of trans-spliced mRNAs. Importantly, in the organisms
under investigation, not all cellular mRNAs are matured through trans-splicing. Thus, cellular mRNAs are generated with the
normal monomethyl cap (MMG-cap) structures as well as trimethyl cap (TMG-cap) structures, the later from SL addition. The
presence of mRNA with two different caps ( m 7 GpppG and m 2,2,7 GpppG) requires that cellular mRNA
cap-binding proteins and mRNA metabolism deal with different cap structures (see Fig. B). A variety of cellular processes are
influenced by the RNA cap structure including nuclear export and import, mRNA translation, and mRNA stability. Proteins that
recognize the cap mediate these processes and therefore play an important role in mRNA metabolism.
A major goal of our current research is to investigate the importance and contribution of the SL
sequence, the different cap structures, and proteins that interact directly or indirectly with the spliced leader and cap on
mRNA metabolism (mRNA translation efficiency, turnover. etc.). In addition, the structural specificity,
affinity, and discrimination of cap-binding and decapping proteins for these 5' terminal elements, their contribution to
mRNA metabolism, and their potential as novel drug targets in parasites are also being studied.
We are exploiting the strengths of several approaches to study the contribution of the SL, mRNA cap,
and the cognate cap-interacting proteins on mRNA metabolism. Competent biochemical extracts for in vitro mRNA
translation and turnover (derived from embryos of the parasitic nematode Ascaris) are being used to dissect the
contribution of both RNA elements and cap-interacting proteins to mRNA translation and turnover. Notably, these competent
biochemical systems are not available in the C. elegans model. RNA biolistics in Ascaris embryos are
being used to study the contribution of trans-spliced elements to mRNA metabolism in vivo. We have also
identified, cloned, and begun to characterize cap-binding proteins from nematodes (Ascaris and C. elegans) including
decapping proteins (DcpS, Dcp1/2), the translation initiation factor eIF4E, and the nuclear cap-binding complex.
Several of these proteins exhibit significant differences in their substrate requirements compared to the human enzymes,
particularly their ability to bind and cleave the trimethylguanosine cap. These cap-interacting proteins represent novel
potential drug targets in parasites of human, veterinary, and agricultural importance. The structure and function of
these novel cap-interacting proteins are currently under investigation as potential rational drug targets. RNA
interference, protein localization, and other studies are also being carried out in C. elegans to better understand the
role of these cap-interacting proteins in gene expression.
2. Development of molecular genetic tools for parasitic helminths (Molecular Parasitology)
Studies on parasitic worms (roundworms and flukes; see medical importance of these organisms below)
have been hampered by a lack of molecular genetic tools including methods for introducing and expressing genes and
altering or knocking out gene function. We recently developed methods to introduce DNA and RNA into parasitic worms
using in vivo biolistics or particle bombardment and electroporation. Another major goal of the lab is to further
develop these and other molecular genetic tools for a variety of experimental approaches (e.g., different parasites, RNA
interference, transgenic parasites, vector development and use, etc.) to examine important aspects of parasite
pathogenesis and biology that could lead to the development of new pharmacological agents against parasitic worms.
Efforts are also currently underway to develop and establish large-scale primary cell cultures from Ascaris
embryos that would enable a variety of new experimental paradigms.
Parasitic Worms
Parasitic worms or helminths remain an important health and economic problem in many parts of the world.
Helminths include blood flukes (schistosomiasis), liver flukes, tapeworms, and a variety of nematodes. Among the nematodes
are Ascaris, hookworms. whipworms and those that cause elephantiasis and river blindness. Nematodes infect 3 billion
people worldwide, leading to considerable morbidity; they are a problem for livestock and domestic animals; and they
result in billions of dollars in annual crop damage. The socioeconomic effects caused by these parasites are severe and
present a major obstacle in facilitating medical and economic improvements in many parts of the world. A major
goal of our work is to develop tools to facilitate the study of worm parasites in an effort to better understand parasite
biology and pathogenesis with the long-term goal of identifying and developing drugs to novel parasite targets.
Selected Publications
Cheng, G., Cohen, L.S., Mikhli, M., Jankowska-Anyszka, M., Stepinski, J., Darzynkiewicz, E., and R.E. Davis. 2007. In vivo translation and stability of trans-spliced mRNAs in nematode embryos. In Press, Mol. Biochem. Parasitol.
Cheng, G, Cohen, L, Ndegwa, D., and R.E. Davis. 2006. The flatworm spliced leader 3' terminal AUG as a translation initiator methionine. J. Biol. Chem. 281:733-743
Lall, S, Piano, F., and R. E. Davis. 2005. C. elegans decapping proteins: Localization and functional analysis of Dcp1, Dcp2, and DcpS during embryogenesis. Mol. Biol. Cell. 16:5880-5890
Cohen, L.S., Mikhli, M., Jiao, X., Kiledjian, M., Kunkel, G., and R.E. Davis. 2005. Dcp2 Decaps m2,2,7GpppN - capped RNAs and its activity is sequence and context dependent. Mol. Cell. Biol. 25:8779-8791.
Kowalska, J., Lewdorowicz, M., Zuberek, J., Bojarska, E., Wojcik, J., Cohen, L.S., Davis, R.E., Stepinski, J., Stolarski, R., Darzynkiewicz, E. and Jemieltity, J. 2005. Synthesis and properties of mRNA cap analogs containing phosphorothioate moiety in 5',5'-triphosphate chain. Nucleosides, Nucleotides & Nucleic Acids. 24:595-600.
Kalek, M., Jemieltity, J., Grudzien, E., Zuberek, J., Bojarska, E., Cohen, L.S., Stepinski, J., Stolarski, R., Davis, R.E., Rhoads, R.E. and Darzynkiewicz, E. 2005. Synthesis and biochemical properties of novel mRNA 5' cap analogs resistant to enzymatic hydrolysis. Nucleosides, Nucleotides & Nucleic Acids. 24:615-621.
Blumenthal, T. and R. E. Davis. 2004. Exploring nematode diversity. Nat. Genet. 12:1246-1247.
Lall, S., Friedman, C., Jankowska-Anyszka, M., Stepinski, J., Darzynkiewicz, E., and R.E. Davis. 2004. Contribution of trans-splicing, 5'-leader length, cap-poly(A) synergism, and initiation factors to nematode translation in an Ascaris suum embryo cell-free system. J. Biol. Chem. 279:45573-45585.
Cohen, L.S., Mikhli, M., Friedman, C., Jankowska-Anyszka, M., Stepinski, J., Darzynkiewicz, E., and R.E. Davis. 2004. Nematode m7GpppG and m32,2,7GpppG Decapping: Activities in Ascaris Embryos and Characterization of C. elegans Scavenger DcpS. RNA 10: 1609-1624.
Higazi T.B., Merriweather, A., Shu,L., Davis, R., and T.R. Unnasch. 2002. Brugia malayi:Transient transfection by microinjection and particle bombardment. Expt. Parasit. 100:95-102.
Davis, R.E., A. Parra, P. LoVerde, E. Ribeiro, G. Glorioso, and S. Hodgson. 1999. Transient expression of RNA and DNA in parasitic helminths by using particle bombardment. Proc. Natl. Acad. Sci. USA 96:8867-8892.
Davis, R.E. and S. Hodgson. 1997. Gene linkage and steady state RNAs suggests trans-splicing may be associated with a polycistronic transcript in the flatworm Schistosoma mansoni. Mol. Biochem. Parasitol. 89:25-39.
Davis, R.E. 1997. Surprising diversity and distribution of SL RNAs in flatworms. Mol. Biochem. Parasitol. 87:29-48.
Davis, R.E. 1996. Spliced leader RNA trans-splicing in metazoa. Parasitology Today 12:33-40.
Davis, R.E., C. Hardwick, P. Tavernier, S. Hodgson, and H. Singh. 1995. RNA trans-splicing in flatworms: Analysis of trans-spliced genes and mRNAs in the human parasite, Schistosoma mansoni. J. Biol. Chem. 270:21813-21819.
Davis, R.E., H. Singh, C. Botka, C. Hardwick, A. Meanawy, and J. Villanueva. 1994. RNA trans-splicing in Fasciola hepatica : Identification of an SL RNA and spliced leader sequences on mRNAs. J. Biol. Chem. 269:20026-20031.
Rajkovic, A., R.E. Davis, J.N. Simonsen and F.M. Rottman. 1990. A spliced leader is present on a subset of mRNAs from the human parasite Schistosoma mansoni. Proc. Natl. Acad. Sci. 87: 8879-8883.
Rajkovic, A., J.N. Simonsen, R.E. Davis and F.M. Rottman. 1989. Molecular cloning and sequence analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase from the human parasite Schistosoma mansoni.. Proc. Natl. Acad. Sci. 86:8217-8221.
Davis, R.E., A.H. Davis, S.M. Carroll, A. Rajkovic and F.M. Rottman. 1988. Tandemly repeated exons encode 81-base repeats in multiple, developmentally regulated Schistosoma mansoni transcripts. Mol. Cell. Biol. 8:4745-4755.
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