Lambowitz Lab - Research

Research in the Lambowitz Lab

Group I introns splice using RNA-catalyzed splicing mechanism. Some group I introns are self-splicing in vitro, however, in most cases proteins are required for efficient splicing in vivo. Our laboratory has indentified several key proteins required for splicing group I introns in Neurospora crassa mitochondria. CYT-18 is a mitochondrial tyrosyl-tRNA synthetase (mt TyrRS) that functions in aminoacylation of mt tRNA^Tyr and in stabilizing the active structure of group I intron RNAs. Biochemical and genetic work along with and a recent co-crystal structure of CYT-18 in complex with a group I intron RNA has identified several insertions within the CYT-18 N-terminal domain that form a distinct group I intron binding site that is non-overlapping with the tRNA binding site. With the structural information now available, CYT-18 provides an excellent model system for studying both the co- evolution of a catalytic RNA and its protein co-factor, and how essential proteins can progressively acquire new functions.

Selected publications:

Paukstelis, P.J., Lambowitz A.M. Identification and evolution of fungal mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity. Proc. Natl. Acad. Sci. 105, 6010-5, 2008.

Paukstelis, P.J., Chen, J-H., Chase, E., Lambowitz, A.M., & Golden, B.L. Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA. Nature 451, 94-97, 2008.

Vicens, Q., Paukstelis P.J., Westhof, E., Lambowitz, A.M., Cech, T.R. Toward predicting self-splicing and protein-facilitated splicing of group I introns. RNA, 14, 2013-29, 2008.

Paukstelis, P.J., Coon, R., Madabusi, L., Nowakowski, J., Monzingo, A., Robertus, J., and Lambowitz, A.M. A tyrosyl-tRNA synthetase adapted to function in group I intron splicing by acquiring a new RNA-binding surface. Mol. Cell, 17, 417-428, 2005.

Caprara, M.G., Mohr, G., and Lambowitz, A.M. A tyrosyl-tRNA synthetase protein induces tertiary folding of the group I intron catalytic core. J. Mol. Biol. 257, 512-531, 1996.

Akins, R.A. and Lambowitz, A.M. A protein required for splicing group I introns in Neurospora mitochondria is mitochondrial tyrosyl-tRNA synthetase or a derivative thereof. Cell, 50, 331-345, 1987.

Mobility and Splicing of Group II Introns

DExH/D-box proteins, commonly referred to as RNA helicases, cause structural transitions in cellular RNPs (e.g. ribosome assembly) by virtue of their ability to unwind RNA duplexes in the presence of nucleotide triphosphates. DExH/D-box proteins have a core motor domain that contains 8 conserved sequence motifs that are involved in ATP binding, hydrolysis, and possibly strand separation. Often the motor domain is flanked by N- and/or C-terminal extensions that are presumed to target the motor domain to a specific site. Genetic studies by us and others identified two DEAD-box (a subfamily of DExH/D-box) proteins involved in mitochondrial (mt) group I and group II intron splicing, CYT-19 in Neurospora crassa and Mss116p in yeast. We have shown that both CYT-19 and Mss116p stimulate the splicing of various group I and group II introns and function in translation and processing of mt RNAs in vivo. Through detailed biochemical analysis in vitro, we have shown that CYT-19 and Mss116p function in splicing by acting as RNA chaperones that bind to RNAs nonspecifically and use their ATP-dependent RNA unwinding activities to resolve kinetic traps in RNA structures. Recently, we have shown that Ded1p, a cytosolic DEAD-box protein from yeast, can stimulate group II intron splicing in vitro.

Selected publications:

Del Campo, M. and Lambowitz, A.M. Structure of the yeast DEAD-box protein Mss116p reveals two wedges that crimp RNA. Mol. Cell 35:598-609, 2009.

Del Campo M, Mohr S, Jiang Y, Jia H, Jankowsky E, Lambowitz AM. Unwinding by local strand separation is critical for the function of DEAD-box proteins as RNA chaperones. J Mol. Biol. 389. 674-93, 2009.

Markov, D.A., Savkina, M., Anikin, M., Del Campo, M., Ecker, K., Lambowitz, A.M., DeGnore, J.P., and McAllister, W.T. Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification. Yeast 26:423-440, 2009.

Del Campo, M. Lambowitz, A.M. 2009. Crystallization and preliminary x-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNP. Acta Crystallogr. F Struct. Biol. Cryst. Commun. 65:832-835, 2009.

Mohr, G., Del Campo, M., Mohr, S., Yang, Q., Jia, H., Jankowsky, E., Lambowitz, A.M. Function of the C-terminal domain of the DEAD-box protein Mss116p analyzed in vivo and in vitro. J. Mol. Biol. 375, 1344-64, 2008.

Del Campo, M., Tijerina, P., Bhaskaran, H., Mohr, S., Yang, Q., Jankowsky, E., Russell, R., Lambowitz, A.M. Do DEAD-box proteins promote group II intron splicing without unwinding RNA?. Mol. Cell. 28, 159-166, 2007.

Mohr, S., Matsuura, M., Perlman, P.S., Lambowitz, A.M. A DEAD-box protein by itself promotes group II intron splicing and reverse splicing by acting as an RNA chaperone. Proc. Natl. Acad. Sci. USA, 103, 3569-3574, 2006.

Mohr, S., Stryker, J., Lambowitz, A.M. A DEAD-box protein functions as an ATP-dependent RNA chaperone in group I intron splicing. Cell, 109, 769-779, 2002.

Mitochondrial Retroplasmids:
Possible Ancestors of Retroviruses

We have found that certain mitochondrial plasmids use a primitive mechanism of reverse transcription that does not require a primer and is analogous to RNA replication. The characteristics of the plasmids suggest they may be related to the early ancestors of retroviruses and possibly to the first DNA elements that emerged at the time of transition from an RNA to DNA world. Studies on the plasmids may provide insight into fundamental aspects of reverse transcription, which is central to retroviral replication.

Selected publications:

Wang H, Lambowitz AM. The Mauriceville plasmid reverse transcriptase can initiate cDNA synthesis de novo and may be related to reverse transcriptase and DNA polymerase progenitor. Cell. 75, 1071-81, 1993.

Wang, H., Kennell, J.C., Kuiper, M.T.R., Sabourin, J.R., Saldanha, R., Lambowitz, A.M. The Mauriceville plasmid of Neurospora crassa. Characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA. Mol. Cell. Biol. 12, 5131-5144, 1992.

Kuiper MT, Lambowitz AM. A novel reverse transcriptase activity associated with mitochondrial plasmids of Neurospora. Cell 55, 693-704, 1988.

	Protein-Dependent Group I Intron Splicing Group I introns splice using RNA-catalyzed splicing mechanism. Some group I introns are self-splicing in vitro, however, in most cases proteins are required for efficient splicing in vivo. Our laboratory has indentified several key proteins required for splicing group I introns in Neurospora crassa mitochondria. CYT-18 is a mitochondrial tyrosyl-tRNA synthetase (mt TyrRS) that functions in aminoacylation of mt tRNA^Tyr and in stabilizing the active structure of group I intron RNAs. Biochemical and genetic work along with and a recent co-crystal structure of CYT-18 in complex with a group I intron RNA has identified several insertions within the CYT-18 N-terminal domain that form a distinct group I intron binding site that is non-overlapping with the tRNA binding site. With the structural information now available, CYT-18 provides an excellent model system for studying both the co- evolution of a catalytic RNA and its protein co-factor, and how essential proteins can progressively acquire new functions. Selected publications: Paukstelis, P.J., Lambowitz A.M. Identification and evolution of fungal mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity. Proc. Natl. Acad. Sci. 105, 6010-5, 2008. Paukstelis, P.J., Chen, J-H., Chase, E., Lambowitz, A.M., & Golden, B.L. Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA. Nature 451, 94-97, 2008. Vicens, Q., Paukstelis P.J., Westhof, E., Lambowitz, A.M., Cech, T.R. Toward predicting self-splicing and protein-facilitated splicing of group I introns. RNA, 14, 2013-29, 2008. Paukstelis, P.J., Coon, R., Madabusi, L., Nowakowski, J., Monzingo, A., Robertus, J., and Lambowitz, A.M. A tyrosyl-tRNA synthetase adapted to function in group I intron splicing by acquiring a new RNA-binding surface. Mol. Cell, 17, 417-428, 2005. Caprara, M.G., Mohr, G., and Lambowitz, A.M. A tyrosyl-tRNA synthetase protein induces tertiary folding of the group I intron catalytic core. J. Mol. Biol. 257, 512-531, 1996. Akins, R.A. and Lambowitz, A.M. A protein required for splicing group I introns in Neurospora mitochondria is mitochondrial tyrosyl-tRNA synthetase or a derivative thereof. Cell, 50, 331-345, 1987.

	Mobility and Splicing of Group II Introns Group II introns are autocatalytic introns that are believed to be related to the progenitors of nuclear pre-mRNA introns in higher organisms. Remarkably, some group II introns are mobile genetic elements that encode reverse transcriptases and insert site-specifically into DNA target sites. We have found that group II intron mobility occurs by a novel retrotransposition mechanism in which the intron RNA inserts directly into a DNA target site and is then reverse transcribed by the intron-encoded protein. The DNA target site is recognized by an RNP complex consisting of the intron-encoded protein and the excised intron RNA, with most of the target site recognized by base pairing of the intron RNA and only a small number of positions recognized by the protein. As a result, group II introns can be programmed to insert into any desired DNA target simply by modifying the intron RNA. Targetron vectors based on mobile group II introns are now being used for genetic engineering and functional genomics of both Gram-negative and Gram-positive bacteria, including commercially and medically important species that lack good genetic systems. In addition, group II introns can be retargeted to insert into medically relevant DNA target sites, such as HIV-1 provirus and the human gene encoding the HIV-1 coreceptor CCR5. We are now attempting to develop targetrons for use in higher organisms, including human cells, with potential applications in gene therapy. Selected publications: Zhuang, F., Matroianni, M., White, T., Lambowitz, A.M. Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joing for cDNA ligation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2009. Dai, L., Chai, D., Gu, S.Q., Gabel, J., Noskov, S.Y., Blocker, F.J., Lambowitz, A.M., Zimmerly, S. A three-dimensional model of a group II intron RNA and its interaction with the intron-encoded reverse transcriptase. Mol. Cell. 30, 472-85, 2008. Mastroianni, M., Watanabe, K., White, T.B., Zhuang, F., Vernon, J., Matsuura, M., Wallingford, J., Lambowitz, A.M. Group II intron-based gene targeting reactions in eukaryotes. PLoS ONE., 3, e3121, 2008. Zhao, J., Niu, W., Yao, J., Mohr, S., Marcotte, E.M., Lambowitz, A.M. Group II intron protein localization and insertion sites are affected by polyphosphate. PLoS Biol. 6, e150, 2008. Pyle, A.M., and Lambowitz, A.M. Group II introns: ribozymes that splice RNA and invade DNA. In The RNA World, 3rd Edition (R.F. Gesteland, T.R. Cech, and J.F. Atkins, Editors), Cold Spring Harbor Laboratory Press, pp. 469-505, Cold Spring Harbor, New York, 2006. Lambowitz, A.M., and Zimmerly, S. Mobile group II introns. Annu. Rev. Genet. 38, 1-35, 2004. Perutka, J., Wang, W., Goerlitz, D., and Lambowitz, A.M. Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes. J. Mol. Biol., 336, 421-439, 2004. Guo, H., Karberg, M., Long, M., Jones, J.P. III, Sullenger, B., and Lambowitz, A.M. Group II introns designed to insert into therapeutically-relevant DNA target sites in human cells. Science, 289, 452-457, 2000 .

	DEAD-Box Proteins in RNA Splicing DExH/D-box proteins, commonly referred to as RNA helicases, cause structural transitions in cellular RNPs (e.g. ribosome assembly) by virtue of their ability to unwind RNA duplexes in the presence of nucleotide triphosphates. DExH/D-box proteins have a core motor domain that contains 8 conserved sequence motifs that are involved in ATP binding, hydrolysis, and possibly strand separation. Often the motor domain is flanked by N- and/or C-terminal extensions that are presumed to target the motor domain to a specific site. Genetic studies by us and others identified two DEAD-box (a subfamily of DExH/D-box) proteins involved in mitochondrial (mt) group I and group II intron splicing, CYT-19 in Neurospora crassa and Mss116p in yeast. We have shown that both CYT-19 and Mss116p stimulate the splicing of various group I and group II introns and function in translation and processing of mt RNAs in vivo. Through detailed biochemical analysis in vitro, we have shown that CYT-19 and Mss116p function in splicing by acting as RNA chaperones that bind to RNAs nonspecifically and use their ATP-dependent RNA unwinding activities to resolve kinetic traps in RNA structures. Recently, we have shown that Ded1p, a cytosolic DEAD-box protein from yeast, can stimulate group II intron splicing in vitro. Selected publications: Del Campo, M. and Lambowitz, A.M. Structure of the yeast DEAD-box protein Mss116p reveals two wedges that crimp RNA. Mol. Cell 35:598-609, 2009. Del Campo M, Mohr S, Jiang Y, Jia H, Jankowsky E, Lambowitz AM. Unwinding by local strand separation is critical for the function of DEAD-box proteins as RNA chaperones. J Mol. Biol. 389. 674-93, 2009. Markov, D.A., Savkina, M., Anikin, M., Del Campo, M., Ecker, K., Lambowitz, A.M., DeGnore, J.P., and McAllister, W.T. Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification. Yeast 26:423-440, 2009. Del Campo, M. Lambowitz, A.M. 2009. Crystallization and preliminary x-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNP. Acta Crystallogr. F Struct. Biol. Cryst. Commun. 65:832-835, 2009. Mohr, G., Del Campo, M., Mohr, S., Yang, Q., Jia, H., Jankowsky, E., Lambowitz, A.M. Function of the C-terminal domain of the DEAD-box protein Mss116p analyzed in vivo and in vitro. J. Mol. Biol. 375, 1344-64, 2008. Del Campo, M., Tijerina, P., Bhaskaran, H., Mohr, S., Yang, Q., Jankowsky, E., Russell, R., Lambowitz, A.M. Do DEAD-box proteins promote group II intron splicing without unwinding RNA?. Mol. Cell. 28, 159-166, 2007. Mohr, S., Matsuura, M., Perlman, P.S., Lambowitz, A.M. A DEAD-box protein by itself promotes group II intron splicing and reverse splicing by acting as an RNA chaperone. Proc. Natl. Acad. Sci. USA, 103, 3569-3574, 2006. Mohr, S., Stryker, J., Lambowitz, A.M. A DEAD-box protein functions as an ATP-dependent RNA chaperone in group I intron splicing. Cell, 109, 769-779, 2002.

	Mitochondrial Retroplasmids: Possible Ancestors of Retroviruses We have found that certain mitochondrial plasmids use a primitive mechanism of reverse transcription that does not require a primer and is analogous to RNA replication. The characteristics of the plasmids suggest they may be related to the early ancestors of retroviruses and possibly to the first DNA elements that emerged at the time of transition from an RNA to DNA world. Studies on the plasmids may provide insight into fundamental aspects of reverse transcription, which is central to retroviral replication. Selected publications: Wang H, Lambowitz AM. The Mauriceville plasmid reverse transcriptase can initiate cDNA synthesis de novo and may be related to reverse transcriptase and DNA polymerase progenitor. Cell. 75, 1071-81, 1993. Wang, H., Kennell, J.C., Kuiper, M.T.R., Sabourin, J.R., Saldanha, R., Lambowitz, A.M. The Mauriceville plasmid of Neurospora crassa. Characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA. Mol. Cell. Biol. 12, 5131-5144, 1992. Kuiper MT, Lambowitz AM. A novel reverse transcriptase activity associated with mitochondrial plasmids of Neurospora. Cell 55, 693-704, 1988.