Crystal structure of the Rna14–Rna15 complex

Abstract

A large protein machinery is required for 39-end processing of mRNA precursors in eukaryotes. Cleavage factor IA (CF IA), a complex in the 39-end processing machinery in yeast, contains four subunits, Rna14, Rna15, Clp1, and Pcf11. Rna14 has a HAT (half a TPR) domain at the N terminus and a region at the C terminus that mediates interactions with Rna15. Rna15 contains a RNA recognition module (RRM) at the N terminus, followed by a hinge region. These two proteins are homologs of CstF-77 and CstF-64 in the cleavage stimulation factor (CstF) of the mammalian 39-end processing machinery. We report the first crystal structure of Rna14 in complex with the hinge region of Rna15, and the structures of the HAT domain of Rna14 alone in two different crystal forms. The complex of the C-terminal region of Rna14 with the hinge region of Rna15 does not have strong interactions with the HAT domain of Rna14, and this complex is likely to function independently of the HAT domain. Like CstF-77, the HAT domain of Rna14 is also a tightly associated dimer with a highly elongated shape. However, there are large variations in the organization of this dimer among the Rna14 structures, and there are also significant structural differences to CstF-77. These observations suggest that the HAT domain and especially its dimer may have some inherent conformational variability.

Structural and biochemical analysis of the assembly and function of the yeast pre-mRNA 3′ end processing complex CF I

“…Unfortunately, crystallography has not been forthcoming with this complex, and EM studies still rely on low resolution approaches (24). Although the Rna14 and Rna15 complex has been studied biophysically (20) and by X-ray crystallography (25) and NMR (19), there is no complete structure with Hrp1. These considerations led us to investigate the complex of Rna14, Rna15, Hrp1, and pre-mRNA using NMR.…”

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

“…With the dimerization of Rna14, they constitute a ␣ 2 ␤ 2 tetramer in the shape of a kinked rod (92). The complex structure of the Rna14 hinge domain and the Rna15 CTD has been obtained alone (91) and together with the Rna14 HAT domain (87) (Fig. 2C).…”

“…Similar characteristics have also been observed in the fungal homolog of CstF-77, Rna14. The HAT domain of Rna14 from Kluyveromyces lactis dimerizes in mostly the same way as that of CstF-77, despite significant structural variations (87). Disruption of this dimerization in yeast can severely impair polyadenylation (88).…”

“…The C-terminal region of CstF-77 contains a proline-rich region that is required for binding to CstF-64 (35,84,90). In yeast, Rna14 and Rna15 (CstF-64 homolog, see below) assemble through the same regions (85,87,91). With the dimerization of Rna14, they constitute a ␣ 2 ␤ 2 tetramer in the shape of a kinked rod (92).…”

The C terminus of Pcf11 forms a novel zinc-finger structure that plays an essential role in mRNA 3′-end processing

“…In yeast, these factors are organized into three complexes called cleavage factor IA (CF IA), cleavage factor II (CF II), and polyadenylation factor I (PF I). The structural basis for the activities of many individual proteins has been characterized (Noble et al 2005(Noble et al , 2007Paulson and Tong 2012;Xiang et al 2014), but how these activities are integrated into the complete processing apparatus and how different steps are orchestrated and coordinated remain unclear at the molecular level.…”