An Arabidopsis Fip1 Homolog Interacts with RNA and Provides Conceptual Links with a Number of Other Polyadenylation Factor Subunits

Abstract

The protein Fip1 is an important subunit of the eukaryotic polyadenylation apparatus, since it provides a bridge of sorts between poly(A) polymerase, other subunits of the polyadenylation apparatus, and the substrate RNA. In this study, a previously unreported Arabidopsis The polyadenylation of messenger RNAs in the nucleus is an important step in the biogenesis of mRNAs in eukaryotes. This RNA processing reaction adds an essential cis element, the poly(A) tail, to the 3Ј-end of a processed pre-mRNA. This process is also coupled with many other steps in mRNA biogenesis (1). Thus, some polyadenylation factors are associated with transcription factors and recruit parts of the polyadenylation apparatus to the transcription initiation complex (2). Polyadenylation is linked to pre-mRNA splicing in a number of ways. For example, interactions between the polyadenylation and splicing machineries are important for the definition of 3Ј-terminal exons in animal cells (3, 4). Other interactions help to modulate different processing fates for pre-mRNAs, thus contributing to the scope of alternative splicing and polyadenylation in eukaryotes. The polyadenylation apparatus interacts with the C-terminal domain of the large subunit of RNA polymerase II (5-9) and with factors that play roles in transcription termination (10); these interactions suggest a central role for 3Ј-end processing in the termination of transcription by RNA polymerase II and subsequent recycling of polymerase II for new rounds of initiation.Polyadenylation is mediated by a multifactor complex in yeast and mammals. This complex recognizes the polyadenylation signal in the pre-mRNA, cleaves the pre-mRNA at a site that is defined by the cis elements, and adds a defined tract of poly(A) to the processed pre-mRNA. In mammals, the factors involved in this process have been classified according to chromatographic and biochemical behaviors, and termed cleavage and polyadenylation specificity factor (CPSF), 2 cleavage-stimulatory factor (CstF), and cleavage factors I and II (CFIm and CFIIm, respectively) (1). Each of these factors in turn consists of several distinct subunits. With the exception of CFIm (the two subunits of which are not obviously apparent in the yeast proteome), yeast possesses a similar array of polyadenylation factor subunits that form a somewhat different set of chromatographically distinct factors, namely cleavage and polyadenylation factor and cleavage factor I (1). Interestingly, the enzyme that adds poly(A) (poly(A) polymerase, or PAP) is part of the cleavage and polyadenylation factor in yeast nuclear extracts but fractionates largely as a separate protein in mammalian extracts. Whereas there are differences in the chromatographic behaviors of the complexes in mammals and yeast, most of the functions of the individual subunits seem to be similar. Besides the PAPs, this includes RNA binding by CPSF160, CPSF30, and CstF64 and their yeast counterparts (Yhh1p, Yth1p, and Rna15p, respectively) (11-17) and bridging between factors (CstF77 and it...

A flexible linker region in Fip1 is needed for efficient mRNA polyadenylation

“…Our data suggest that the attachment point of Pap1 to Fip1 is not as important in the cell as the recruitment of Pap1 onto the RNA, which allows efficient initiation of polyadenylation. This is consistent with our simplified CPF model and with the mammalian and plant systems, where Fip1 contains RNA-binding domains (Kaufmann et al 2004;Forbes et al 2006. )…”

“…This interaction between Fip1 and CF IA is conserved across species. CstF 77, which is the homolog of the Rna14 subunit of CF IA, interacts with Fip1 in humans (Kaufmann et al 2004), and plants (Forbes et al 2006;Addepalli and Hunt 2008). Unlike the Fip1/Yth1 interaction, these contacts are not needed to hold Fip1/Pap1 in the CPF complex (Fig.…”

“…The interaction between both proteins is conserved in humans and plants (Kaufmann et al 2004;Forbes et al 2006), but the atomic details of this interaction are not (Meinke et al 2008). Fip1 in turn is held in the 39 end complex through its interaction with Yth1 (Barabino et al 2000).…”

Delineating the Structural Blueprint of the Pre-mRNA 3′-End Processing Machinery

“…Mutations that disrupt this interaction cause cell death (72). Interestingly, residues at the interface in both Fip1 and Pap1 are not conserved but the interaction between these two proteins has been observed across different species (71)(72)(73), suggesting that the tethering but not the atomic details of the interaction is essential for polyadenylation (74).…”

Unique Features of Plant Cleavage and Polyadenylation Specificity Factor Revealed by Proteomic Studies

“…These should be considered as factors located at the periphery of the CPSF complex or factors belonging to other polyadenylation complexes but that still closely interact with CPSF. Among them, AtFIPS5 only exists in the protein pool copurified with AtCPSF30 (Table I, [B]), and its only interacting partner identified by Y2H study was also AtCPSF30 (Table I, [b]; Forbes et al, 2006). Moreover, in mammals, CPSF30 and hFip1 belong to the same CPSF complex (Zhao et al, 1999), and their yeast homologs, Yth1p and Fip1p, exist in the same polyadenylation complex, PF-I (Mandel et al, 2008).…”