RRM
2009 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
The 77 kDa subunit of the heterotrimeric complex known as the cleavage stimulation factor (CstF-77) plays an integral role in messenger RNA 3’end processing. Previous studies have revealed the C-terminus of the Arabidopsis ortholog of CstF-77 (AtCstF-77) exhibits protein-protein interactions with the N-terminus of AtCstF-64 and an Arabidopsis Fip1 (Factor Interacting with Poly-A polymerase) ortholog (1,2). In addition, it has been demonstrated through yeast two-hybrid analyses that AtCstF-77 interacts with the Arabidopsis ortholog of the 30 kD subunit of the Cleavage and Polyadenylation Specificity Factor (AtCPSF-30) (3). To further understand this latter interaction, the crystal structure of mammalian CstF-77 was used to appropriately divide AtCstF-77 into three regions: the N-terminus, Middle, and C-terminus (4). In vitro pull-down assays were carried out with each region to determine the region(s) binding AtCPSF-30. These experiments demonstrated that that the C-terminus of At-CstF-77 interacts with AtCPSF-30, confirming previous studies. As AtCPSF-30 is an RNA-binding protein, the effects of AtCstF-77 on RNA binding by AtCPSF-30 were assayed. Remarkably, in the course of these studies, it was found that the C-terminus of AtCstF-77 by itself possesses RNA-binding activity. This activity seemed non-specific in terms of RNA sequence requirements since RNAs lacking the plant polyadenylation signal sub-elements (near-upstream, far-upstream, and cleavage elements) were all equally effective in binding to AtCstF-77. These studies therefore reveal AtCstF-77 to be an RNA binding protein. This adds yet another RNA-binding activity to the plant polyadenylation complex, and raises interesting questions as to the means by which RNAs are recognized and handled in the course of mRNA 3’ end formation in plants.
References:
1. Yao Y, Song L, Katz Y, Galili G: Cloning and characterization of Arabidopsis homologues of the animal CstF complex that regulates 3\' mRNA cleavage and polyadenylation. J Exp Bot 2002, 53:2277-2278.
2. Forbes KP, Addepalli B, Hunt AG: An Arabidopsis Fip1 homolog interacts with RNA and provides conceptual links with a number of other polyadenylation factor subunits. J Biol Chem 2006, 281:176-186.
3. Hunt AG, Xu R, Adepalli B, Rao S, Forbes KP, Meeks LR, Xing D, Mo M, Zhao H, Bandyopadhyay A, Dampanaboina L, Marion A, Von Lanken C, Li QQ: Arabidopsis mRNA polyadenylation machinery: a comprehensis analysis of protein-protein interactions and gene expression profiling. BMC Genomics 2008, 9:220.
4. Bai Y. Auperin TC, Chou CY, Chang GG, Manley J, Tong L: Crystal Structure of Murine CstF-77: Dimeric Association and Implications for Polyadenylation of mRNA Precursors. Molecular Cell 2007, 25: 863-875.
Keywords: polyadenylation, RNA-binding, CstF-77, and CPSF-30
