Reconstitution of CF IA from Overexpressed Subunits Reveals Stoichiometry and Provides Insights into Molecular Topology

Gordon, James M.; Shikov, Sergei; Kuehner, Jason N.; Liriano, Melissa A.; Lee, Eun-Hee; Stafford, Walter F.; Poulsen, Mathias Bach; Harrison, Celia J.; Moore, Claire; Bohm, Andrew

doi:10.1021/bi200964p

Cited by 32 publications

(38 citation statements)

References 48 publications

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“…Our structure of the Rna14 HAT domain dimer is supported by published mutagenesis data on S. cerevisiae Rna14 (Gordon et al 2011), which showed that the R562E/Y563S double mutation disrupted its dimerization. These two residues are equivalent to Lys565 and Tyr566 of K. lactis Rna14 and are located in the center of the HAT domain dimer interface.…”

Section: Resultssupporting

confidence: 78%

“…This suggests that the linker between the HAT domain and the region at the C terminus of Rna14 that interacts with Rna15 is accessible to proteases and may be flexible. The flexibility of this linker has also been reported for the S. cerevisiae Rna14-Rna15 complex (Gordon et al 2011). The crystal structure of K. lactis Rna14 in crystal form 1 was determined at 2.8 Å resolution by the selenomethionyl single-wavelength anomalous diffraction (SAD) method (Hendrickson 1991).…”

Section: Resultsmentioning

confidence: 96%

“…These two residues are equivalent to Lys565 and Tyr566 of K. lactis Rna14 and are located in the center of the HAT domain dimer interface. The mutation reduced both the affinity of the Rna14-Rna15 complex for binding RNA and the activity of the 39-end processing machinery for cleavage and polyadenylation of pre-mRNA (Gordon et al 2011), demonstrating the functional importance of the dimerization of Rna14.…”

Section: Resultsmentioning

confidence: 99%

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Crystal structure of the Rna14–Rna15 complex

Paulson

Tong²

2012

RNA

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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.

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Section: Resultssupporting

confidence: 78%

Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

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Crystal structure of the Rna14–Rna15 complex

Paulson

Tong²

2012

RNA

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“…To better understand how 3′-end processing signals are recognized to initiate 3′-end processing and transcription termination, we sought to obtain structural information on the complete CF I complex including Rna14, Rna15, Hrp1, and the pre-mRNA transcript. 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.…”

Section: Resultsmentioning

confidence: 99%

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

Barnwal

Lee

Moore

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The accuracy of the 3′-end processing by cleavage and polyadenylation is essential for mRNA biogenesis and transcription termination. In yeast, two poorly conserved neighboring elements upstream of cleavage sites are important for accuracy and efficiency of this process. These two RNA sequences are recognized by the RNA binding proteins Hrp1 and Rna15, but efficient processing in vivo requires a bridging protein (Rna14), which forms a stable dimer of heterodimers with Rna15 to stabilize the RNA-protein complex. We earlier reported the structure of the ternary complex of Rna15 and Hrp1 bound to the RNA processing element. We now report the use of solution NMR to study the interaction of Hrp1 with the Rna14-Rna15 heterodimer in the presence and absence of 3′-end processing signals. By using methyl selective labeling on Hrp1, in vivo activity and pull-down assays, we were able to study this complex of several hundred kDa, identify the interface within Hrp1 responsible for recruitment of Rna14 and validate the functional significance of this interaction through structure-driven mutational analysis.mRNA processing | mRNA transport | protein-RNA | methyl-TROSY | modeling E ukaryotic messenger RNA precursors (pre-mRNA) undergo extensive cotranscriptional processing before their transport to the cytoplasm and subsequent translation (1, 2). The processing events include 5′-end capping, splicing to remove intronic sequences and cleavage and polyadenylation at the 3′-end (3, 4). Correct 3′-end processing is necessary for mRNA biogenesis and transcription termination, preserves RNAs from degradation (5), promotes its export to the cytoplasm (6), enhances translation (7) and promotes transcriptional activation (4, 8). Cleavage and polyadenylation can be uncoupled in vitro but in vivo they are connected with each other and with transcription termination. Defects in 3′-end processing have also been associated with several diseases in humans (9), but this process may be even more critical in yeast that has shorter intergenic regions and much rarer alternative splicing (10, 11). In this organism, efficient mRNA transport is mostly dependent on polyadenylation rather than splicing.In yeast, the 3′-end processing reaction is executed by a complex machine containing more than 20 proteins (4) which share significant similarities with human 3′-end processing proteins. The entire yeast 3′-end processing complex is divided into two subcomplexes, called cleavage factor I (CF I) and cleavage and polyadenylation factor (CPF) (4). CF I binds to the pre-mRNA upstream of the cleavage signal within the pre-mRNA, whereas CPF binds at the cleavage site as well as upstream and downstream of the cleavage site. Several sequences within yeast pre-mRNAs, which are surprisingly poorly conserved, direct the recruitment of cleavage and polyadenylation factors (12, 13). In the 5′-3′ direction, they include AU-rich efficiency element (EE) responsible for polyadenylation efficiency; A-rich positioning element (PE) critical for precise 3′-end processing; t...

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“…According to the BioGRID database, there are seven interacting partners for Pcf11 identified by at least two experimental approaches, although some of these interactions might be mediated by another factor: Clp1, Rna14, Rna15, Rpo21, Ysh1, Ydh1, and Yra1. The middle region of Pcf11 helps in the assembly and localization of Rna14/Rna15 and Clp1 to the 3 ′ -UTR (Noble et al 2007;Qu et al 2007;Gordon et al 2011). Yra1 putatively interacts with Pcf11 through a region N-terminal to the very C-terminal zinc finger to facilitate assembly of the pre-mRNA for recognition by the RNA export machinery (Johnson et al 2009(Johnson et al , 2011.…”

Section: Discussionmentioning

confidence: 99%

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

Yang

Hsu

Lee

et al. 2016

RNA

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3′ -End processing of pre-mRNAs prior to packaging and export to the cytoplasm of the mature transcript is a highly regulated process executed by several tens of protein factors that recognize poorly conserved RNA signals. Among them is Pcf11, a highly conserved, multidomain protein that links transcriptional elongation, 3′ -end processing, and transcription termination. Here we report the structure and biochemical function of Pcf11's C-terminal domain, which is conserved from yeast to humans. We identify a novel zinc-finger fold, resembling a trillium flower. Structural, biochemical, and genetic analyses reveal a highly conserved surface that plays a critical role in both cleavage and polyadenylation. These findings provide further insight into this important protein and its multiple functional roles during cotranscriptional RNA processing.

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Reconstitution of CF IA from Overexpressed Subunits Reveals Stoichiometry and Provides Insights into Molecular Topology

Cited by 32 publications

References 48 publications

Crystal structure of the Rna14–Rna15 complex

Crystal structure of the Rna14–Rna15 complex

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

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

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