Protein-facilitated group II intron splicing

Catalytic ribonucleoprotein particles (RNPs) are at the core of several fundamental cellular processes, including protein synthesis, tRNA processing, and RNA splicing. The contributions of the RNA and protein components are varied. In some instances, RNA subunits harbor catalytic activity and the proteins enhance the formation or stability of the active RNA structure. In others, RNAs provide an assembly scaffold for catalytically-active proteins. RNA and protein molecules are known to cooperate in forming substrate binding surfaces and, in principle, they could cooperate to form an active site. We are using RNPs harboring "self-splicing" group II introns to explore modes of cooperation between RNA and protein. Group II introns are large, highly structured catalytic RNAs that require proteins to function efficiently in vivo. The chemical mechanism of group II intron splicing is identical to that in the spliceosome and it is speculated that these two splicing machineries evolved from a common ancestor. Group II intron RNPs are excellent models for dissecting the roles of RNA and protein subunits in RNP function because they are relatively simple, and because proteins that facilitate intron function can be identified genetically and then studied in vitro.

We have identified nine nucleus-encoded proteins that are required for the splicing of various subsets of the 17 group II introns in maize chloroplasts (see Figure) and we have shown that each protein is associated with its intron substrates in stable RNPs in vivo. Currently we are dissecting the biochemical interactions betweenthese proteins and their cognate introns to elucidate how each protein contributes to the splicing process.

Publications

Asakura Y and Barkan A. (2007) A CRM domain protein functions dually in group I and group II intron splicing in land plant chloroplasts. Plant Cell, in press.

Watkins KP, Kroeger TS, Cooke AM, Williams-Carrier RE, Friso G, Belcher SE, van Wijk KJ, and Barkan A. (2007) A Ribonuclease III Domain Protein Functions in Group II Intron Splicing in Maize Chloroplasts. Plant Cell, 19: 2606-2623

Asakura Y and Barkan A. (2006) Arabidopsis orthologs of maize chloroplast splicing factors promote splicing of orthologous and species-specific group II introns. Plant Phys., 142: 1656-1663.

Schmitz-Linneweber C, Williams-Carrier R, Williams-Voelker PM, Kroeger TS, Vichas A, and Barkan A. (2006) A pentatricopeptide repeat protein facilitates the trans-splicing of the maize chloroplast rps12 pre-mRNA. Plant Cell, 18:2650-63.

Ostheimer GJ, Rojas M, Hadjivassiliou H, and Barkan A. (2006) Formation of the CRS2-CAF2 group II intron splicing complex is mediated by a 22 amino acid motif located in the C-terminal region of CAF2. J. Biol. Chem., 281: 4732-8

Schmitz-Linneweber C, Williams-Carrier R, Barkan A. (2005) RNA Immunoprecipitation and Microarray Analysis Show a Chloroplast Pentatricopeptide Repeat Protein to Be Associated with the 5' Region of mRNAs Whose Translation It Activates The Plant Cell 17: 2791-2804

Ostersetzer O., A.M. Cooke, K.P. Watkins, and A. Barkan. (2005) CRS1, a Chloroplast Group II Intron Splicing Factor, Promotes Intron Folding through Specific Interactions with Two Intron Domains. Plant Cell 17: 241-55.

Ostheimer G.J., H. Hadjivasiliou, D.P. Kloer, A. Barkan, and B.W. Matthews. (2005) Structural analysis of the group II intron splicing factor CRS2 yields insights into its protein and RNA interaction surfaces. J Mol Biol 345: 51-68.

Ostheimer G.J., R. Williams-Carrier, S. Belcher, E. Osborne, J. Gierke, and A. Barkan (2003) Group II intron splicing factors derived by duplication and diversification of an ancient RNA binding domain. EMBO J 22: 3919-29.

Ostheimer G.J., A. Barkan, and B.W. Matthews. (2002) Crystal Structure of E. coli YhbY. A representative of a novel class of RNA-binding proteins. Structure 10: 1593-1601.

Till B., C. Schmitz-Linneweber, R. Williams-Carrier, and A. Barkan (2001) CRS1 is a novel group II intron splicing factor that was derived from a domain of ancient origin. RNA 7:1227-38.

Jenkins, B.D. and A. Barkan (2001) Recruitment of a peptidyl-tRNA hydrolase as a facilitator of group II intron splicing in chloroplasts. EMBO J 20:872-9.

Jenkins, B., Kulhanek, D., and Barkan, A. (1997) Nuclear mutations that block group II RNA splicing in maize chloroplasts reveal several intron classes with distinct requirements for splicing factors. Plant Cell, 9: 283-296.

Barkan A (1989) Tissue-dependent plastid RNA splicing in maize: transcripts from four plastid genes are predominantly unspliced in leaf meristems and roots. The Plant Cell 1: 437-445.