Original Article

Cell Research (2011) 21:1039-1051. doi:10.1038/cr.2011.67; published online 12 April 2011

Structural basis of pre-mRNA recognition by the human cleavage factor Im complex

Heng Li1,2, Shuilong Tong1,2, Xu Li1,2, Hui Shi1,2, Zheng Ying1, Yongxiang Gao1,2, Honghua Ge1,2, Liwen Niu1,2 and Maikun Teng1,2

  1. 1Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China
  2. 2Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei 230026, China

Correspondence: Maikun Teng, Tel: 86-551-3606314; Fax: 86-551-3601443
E-mail: mkteng@ustc.edu.cn; Liwen Niu, E-mail: lwniu@ustc.edu.cn

Received 3 August 2010; Revised 25 October 2010; Accepted 17 December 2010; Published online 12 April 2011.

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Abstract

The cleavage factor Im (CF Im), consists of a 25 kDa subunit (CF Im25) and one of three larger subunits (CF Im59, CF Im68, CF Im72), and is an essential protein complex for pre-mRNA 3′-end cleavage and polyadenylation. It recognizes the upstream sequence of the poly(A) site in a sequence-dependent manner. Here we report the crystal structure of human CF Im, comprising CF Im25 and the RNA recognition motif domain of CF Im68 (CF Im68RRM), and the crystal structure of the CF Im-RNA complex. These structures show that two CF Im68RRM molecules bind to the CF Im25 dimer via a novel RRM-protein interaction mode forming a heterotetramer. The RNA-bound structure shows that two UGUAA RNA sequences, with anti-parallel orientation, bind to one CF Im25-CF Im68RRM heterotetramer, providing structural basis for the mechanism by which CF Im binds two UGUAA elements within one molecule of pre-mRNA simultaneously. Point mutation and kinetic analyses demonstrate that CF Im68RRM can bind the immediately flanking upstream region of the UGUAA element, and CF Im68RRM binding significantly increases the RNA-binding affinity of the complex, suggesting that CF Im68 makes an essential contribution to pre-mRNA binding.

Keywords:

cleavage factor Im (CF Im); pre-mRNA processing; poly(A) site recognition; RRM domain; RNA binding

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Introduction

Eukaryotic pre-mRNAs are synthesized and post-transcriptionally modified in the nucleus, before being exported into the cytoplasm to serve as templates for protein synthesis. The post-transcriptional modifications comprise 5′-end capping, splicing and 3′-end formation of the pre-mRNA. The maturation of the 3′-ends of most mRNA is catalyzed by multiple protein complexes, and requires the endonucleolytic cleavage of primary transcripts and the addition of poly(A) tails to the upstream cleavage products.

In mammals, the factors that are required for mRNA maturation in vitro include the cleavage and polyadenylation specificity factor (CPSF), cleavage stimulatory factor (CstF), cleavage factors Im and IIm (CF Im and CF IIm), poly(A) polymerase (PAP) and nuclear poly(A) binding protein (PABN2). The cleavage reaction requires CPSF, CstF, CF Im, CF IIm, and PAP. CPSF binds the highly conserved AAUAAA hexamer upstream of the cleavage site, and CstF binds the GU/U-rich sequence downstream of the cleavage site 1. CPSF and CstF interact to form a stable complex before binding the pre-mRNA to recognize the two elements in vivo 2, 3. CF Im binds the pre-mRNA substrate in the vicinity of the poly(A) site concomitantly with CPSF. This stabilizes the binding between CPSF and the AAUAAA hexamer, facilitating pre-mRNA 3′-end processing complex assembly, and therefore enhances the rate and overall efficiency of poly(A) site cleavage in vitro 4, 5, 6. Sequence-specific binding of CF Im to pre-mRNA directs A(A/U)UAAA-independent poly(A) addition through interaction with the poly(A) polymerase and a CPSF subunit, hFip1 7. After cleavage, CPSF remains bound to the upstream cleavage fragment, and recruits PAP onto the 3′-end of pre-mRNA. It also cooperates with PABN2 in the addition of a 250-nucleotide long poly(A) tail to the upstream cleavage fragment 8. SELEX analysis has shown that CF Im recognizes a five-nucleotide element, UGUAN (N = A > U ≥ C/G) with high affinity 7. When added to partially purified 3′-end processing factors, recombinant CF Im is sufficient to reconstitute poly(A) site cleavage activity in vitro (the CF Im complex used in this study was a CF Im25-CF Im68 complex, as discussed further below) 5. Repression of CF Im activity by knocking down CF Im25 does not affect HeLa cell viability, but increases the usage of the upstream poly(A) site, suggesting that CF Im25 has an important role in poly(A) site selection 9.

CF Im has been characterized as a heterodimer, consisting of a 25 kDa subunit (CF Im25) and one of three larger subunits (CF Im59, CF Im68 or CF Im72) 5. CF Im25, which is also known as NUDT21 or CPSF5, is a 227-amino acid polypeptide, which is highly conserved in metazoan, and which contains a nucleoside diphosphate linked to some other moiety, x (NUDIX) hydrolase domain (residues 79-203) 10. CF Im68, a member of the SR family of splicing factors, is a 551-amino acid polypeptide, which features an RNA recognition motif (RRM) domain at its N-terminal region, a central proline-rich region, and a C-terminal arginine/serine-rich (RS) domain. The RRM domain, which is also known as a RNA-binding domain (RBD) or ribonucleoprotein domain (RNP), is a motif found commonly in all organisms. It is characterized by an RNP1 consensus sequence (K/R-G-F/Y-G/A-F/Y-V/I/L-X-F/Y) and an RNP2 consensus sequence (V/I/L-F/Y-V/I/L-X-N/L) formed by aromatic and positively charged residues 11, 12, 13. The RS domain is required for protein-protein interactions with other RS domains 1, 14.

In this study, we report the structure of CF Im, comprising CF Im25 (residues 34-227) and the RRM domain of CF Im68 (CF Im68RRM, residues 78-159), and the structure of a CF Im25-CF Im68RRM-RNA complex. The structural and mutational data reveals a novel RRM-protein binding mode, in which two CF Im68RRM molecules bind to a CF Im25 homodimer to form a heterotetramer. The structure of the CF Im-RNA complex shows that two UGUAA RNA sequences, with anti-parallel orientation, bind simultaneously to one CF Im25-CF Im68RRM heterotetramer. Kinetic analyses demonstrate that the complex assembly increases RNA-binding affinity, and subsequent mutagenesis analyses reveal that CF Im68 interacts with the immediately flanking upstream region of the UGUAA element via the L3 loop of the RRM domain of CF Im68.

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Results

CF Im68RRM is sufficient for CF Im25 binding

In an in vitro binding assay, the N-terminal region of CF Im68 (CF Im68N, residues 1-226) has been shown to interact with CF Im25 15. The central and C-terminal regions of CF Im68 (residues 209-551) do not interact with CF Im25 15. We have carried out detailed characterization of the region of CF Im68 responsible for CF Im25 binding. Pull-down assays showed that both CF Im68N and CF Im68RRM bind to GST-CF Im25 (Supplementary information, Figure S1A). As the molecular weight of CF Im68N is similar to that of GST alone, the GST-Rtt106p fusion was used as a negative control in binding studies. We also tested whether the N-terminal extension (RRMN, residues 1-80) or the C-terminal extension (RRMC, residues 160-226) of CF Im68RRM interacts with CF Im25. Immunoblot analysis showed neither His-MBP-RRMN nor His-MBP-RRMC binds to GST-CF Im25 (Supplementary information, Figure S1B). These results demonstrate that the RRM domain of CF Im68 is sufficient for CF Im25 binding.

Overall structure of the CF Im25-CF Im68RRM complex

To better characterize CF Im, we attempted to determine the structure of the CF Im25-CF Im68RRM complex using X-ray crystallography (Table 1). Crystals obtained from the full-length CF Im25 in a complex with CF Im68RRM were not of an adequate quality to allow data collection. In the structure of apo-CF Im25, residues 1-20 were not observed in the electron density map and residues 21-32 formed an extended loop structure 16. To obtain crystals for high-resolution studies, a truncated CF Im25 protein, with residues 1-33 removed, was constructed. A crystal, which diffracted to a resolution of 2.7 Å, was obtained and the structure was subsequently determined (see Materials and Methods for details). The CF Im25-CF Im68RRM complex was found to be a heterotetramer (approximate dimensions of 95 Å × 75 Å × 60 Å), with a pseudodyad passing through the heterotetramer, relating the pair of heterodimers (Figure 1). The heterotetrameric state of the CF Im25-CF Im68RRM complex was confirmed by size-exclusion chromatography, with the complex eluting with an apparent molecular weight of around 61.5 kDa (Supplementary information, Figure S2B and S2C). As the molecular weights of CF Im25 and CF Im68RRM are 24 and 10 kDa, respectively, this result suggests that the CF Im25-CF Im68RRM complex exists as a heterotetramer in solution. The heterotetrameric state is also consistent with previous reports that two subunits of CF Im form a heterotetramer in solution 17.

Figure 1.
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Overall structure of the CF Im25-CF Im68RRM complex. View of the CF Im25-CF Im68RRM complex in two orientations. The lower ribbon diagram of the complex is rotated by 90° around the horizontal axis relative to the upper one. A CF Im25 dimer (yellow and magenta) binds two CF Im68RRM molecules (green and red) forming a heterotetramer.

Full figure and legend (66K)


Structure of CF Im68RRM

Although the sequence identity of CF Im68RRM with other RRM domains is less than 30%, CF Im68RRM adopts the classical compact αβ sandwich structures observed in other RRM domains, with a topology of β1α1β2β3α2β4 (Figure 2A). Residues 80-86 (β1), 110-114 (β2), 127-131 (β3), 155-158 (β4) constitute the four-stranded anti-parallel β-sheet arranged in the order of β4β1β3β2, from left to right, when facing the sheet, and two α helices, the α1 helix (residues 94-104) and the α2 helix (residues 134-146), pack against the β-sheet (Figure 2B). The RNP1 motif (residues 124-KGFALVGV-131) and the RNP2 motif (residues 83-LYIGNL-88) are located in the β3 and β1 strands, respectively. CF Im68RRM has only 26% and 22% sequence identities with the RRM domain of CBP20 18, 19 and the second RRM domain of sex-lethal protein (SXL-RRM2) 20, respectively. However, the program DALI 21 revealed that CF Im68RRM is structurally similar to both CBP20 (Z score = 12.5) and SXL-RRM2 (Z score = 13.1). Superimposition of CBP20 and SXL-RRM2 with CF Im68RRM showed RMS deviations at Cα positions of 1.01 Å with 52 residues and 1.30 Å with 57 residues, respectively, (Figure 2C). Tyr84 and Phe126 in RNP2 and RNP1 motifs of CFIm68RRM are equivalent to Tyr43 and Phe83 in CBP20, respectively, and Tyr84 inCF Im68RRM is equivalent to Tyr214 in SXL-RRM2.

Figure 2.
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Structure of CF Im68RRM. (A) Structure-based alignment of RRM domains. RNP1 and RNP2 are highlighted in cyan and yellow. CF Im68RRM adopts the canonical αβ sandwich structure with a β1α1β2β3α2β4 topology. (B) Structure of CF Im68RRM. Four β-strands constitute an anti-parallel β-sheet arranged in the order of β4β1β3β2, from left to right when facing the sheet, and two α helices pack against the β-sheet. The mutated residues in Figure 6C are indicated by ball-and-stick representation and highlighted. (C) Superimpositions of CF Im68RRM with CBP20 and SXL-RRM2 (Pymol). CF Im68RRM, CBP20 and SXL-RRM2 are colored in green, yellow and magenta, respectively. Tyr84 and Phe126 of CF Im68RRM, Tyr43 and Phe83 of CBP20 and Tyr214 of SXL-RRM2 are indicated by ball-and-stick representation and are highlighted.

Full figure and legend (104K)

Interface between CF Im25 and CF Im68RRM

Complex assembly between the CF Im25 dimer and one molecule of CF Im68RRM buries a surface area of 1 200 Å2 (AREAIMOL 22). The L1 and L3 loops of CF Im68RRM interact with the L10 loop of CF Im25 (Figure 3). This binding interface is strengthened by three hydrogen bonds and by more than 10 hydrophobic contacts. Tyr158, Tyr160 and His164 of CF Im25 form three hydrogen bonds with Arg118, Asn120 and Glu116 of CF Im68RRM, respectively. Tyr158 of CF Im25 forms hydrophobic contacts with the backbone of Ala119, Asn120 and Gly121 of CF Im68RRM, and Ala163 of CF Im25 interacts hydrophobically with the backbone of Trp90 and Trp91 of CF Im68RRM. His164 of CF Im25 forms hydrophobic interactions with the backbone of Ser123 of CF Im68RRM. Compared with the previously published structure of apo-CF Im25 16, the side chain of His164 is rotated by 120° and reaches into a hydrophobic pocket, which is negatively charged at the top but is hydrophobic at the bottom, interacting with many residues of CF Im68RRM (Supplementary information, Figure S3). These interactions fix the L3 loop of CF Im68RRM in an extended conformation. CF Im68RRM also interacts with the second CF Im25 molecule of the homodimer via hydrophobic contacts (Figure 3). The L1 loop of CF Im68RRM interacts with the L5 loop and α4 helix of CF Im25. His89 of CF Im25 interacts with Trp91 of CF Im68RRM, and Phe199 of CF Im25 interacts with Trp90, Asn120 and Gln122 of CF Im68RRM.

Figure 3.
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The interfaces between the CF Im25 dimer and CF Im68RRM. The L10 loop of CF Im25 (yellow) interacts with L1 and L3 loops of CF Im68RRM (green). The L5 loop and α4 helix of the other CF Im25 molecule (magenta) of the dimer interacts with the L1 loop of CF Im68RRM. The residues involved in these contacts are indicated by ball-and-stick representation and are highlighted.

Full figure and legend (58K)

Mutational analysis supports structural model for RRM domain binding

To validate the two interfaces observed in the structure, two mutants of GST-tagged CF Im25 were generated (GST-mutant1 and GST-mutant2). In GST-mutant1, Tyr158, Tyr160 and His164 were substituted and in GST-mutant2, His89 and Phe199 were substituted. Pull-down assays showed that CF Im68RRM efficiently bound to GST-tagged wild-type CF Im25 (GST-wtCF Im25), whereas CF Im68RRM did not bind to either the GST-mutant1 or the GST-mutant2 (Figure 4A). These results suggest that CF Im68RRM interacts with both CF Im25 molecules of the dimer, indicating the importance of CF Im25 dimerization for CF Im68RRM binding.

Figure 4.
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The CF Im25 dimerization is important for CF Im68RRM binding. (A) Coomassie blue-stained protein SDS gel shows that GST-wtCF Im25 can efficiently bind to CF Im68RRM, but GST-mutant1 and GST-mutant2 does not, suggesting that CF Im68RRM interacts with both CF Im25 molecules of the dimer. (B) The other CF Im25 molecule of the dimer hydrophobically interacts with the L10 loop. The L10 loop is shown in magenta and the other CF Im25 molecule is shown in yellow. Residues involved in hydrophobic contacts are indicated by ball-and-stick representation and are highlighted. (C) Immunoblot analysis shows that the wild-type CF Im25 (wtCF Im25) efficiently binds to GST-CF Im68RRM, whereas mtCF Im25 does not. Neither wtCF Im25 nor mtCF Im25 binds to the GST tag.

Full figure and legend (70K)

Although the L10 loop of CF Im25 (residues 151-168) is a tortuous loop, it is well ordered in the apo-CF Im25 structure. Superimposition of the CF Im68RRM-bound CF Im25 structure with the apo structure showed that the L10 loops of CF Im25 adopt almost the same conformation. The conformation of the L10 loop of CF Im25 could be important for CF Im68RRM binding. Detailed structural analyses revealed that Leu91, Phe199, Tyr202, Pro216 and Gln217 of one CF Im25 molecule of the dimer hydrophobically interacts with Tyr158, Ala163, Pro159 and Tyr160 of the L10 loop of the other CF Im25 molecule and that these interactions may stabilize the L10 loop (Figure 4B). Mutagenesis and immunoblot analyses were performed to investigate this hypothesis. A mutant of CF Im25 (mtCF Im25) was generated, in which Leu91, Tyr202, Pro216 and Gln217 were replaced by alanines to eliminate the proposed stabilization of the L10 loop. Size-exclusion chromatography showed that mtCF Im25 eluted similarly to the wild-type CF Im25 (wtCF Im25), indicating that these mutations do not affect dimerization (Supplementary information, Figure S2D). Immunoblot analysis showed that wtCF Im25 bound efficiently to GST-CF Im68RRM, whereas mtCF Im25 did not (Figure 4C). Thus, substitution of four of the five residues (Leu91, Tyr202, Pro216 and Gln217) markedly impairs CF Im68RRM binding, suggesting that conformation of the L10 loop of CF Im25 is possibly important for CF Im68RRM binding.

Structure of the CF Im25-CF Im68RRM-UGUAA complex

We determined the crystal structure of the CF Im25-CF Im68RRM-UGUAA complex (Table 1). The UGUAA element binds to CF Im25 in a positively charged cavity formed by the NUDIX domain (Figure 5A). As the electron density map for the chain S of the UGUAA sequences is the best (Figure 5B), the chain S is taken to describe the interactions between CF Im25 and the UGUAA element. The RNA strand twists by about 90° after U3, flipping A4 out of the binding cavity, and twists back right after A4 (Figure 5A). The main chains of Tyr208 and Gly209 make hydrophobic contacts with the uracil base of U1, whereas Phe103 stabilizes the guanine base of G2 through a base-stacking interaction (Supplementary information, Figure S4A). The N3 and O2 of U1 form hydrogen bonds with the main chain carbonyl and amino groups of Phe104, respectively, (Supplementary information, Figure S4A). The O4 and N3 of U3 form hydrogen bonds with the N2 group of the side chain of Arg63 and main chain carbonyl of Ser58, respectively (Supplementary information, Figure S4A). A5 interacts with Leu99 and Gly100 via hydrophobic contacts (Supplementary information, Figure S4B). The N6 and N1 of A5 form hydrogen bonds with the N3 group of G2 and O4* group of the sugar ring of U3, respectively, (Supplementary information, Figure S4B). A4 does not interact with CF Im25.

Figure 5.
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Structure of the CF Im25-CF Im68RRM-RNA complex. (A) The UGUAA element is located in a positively charged cavity of CF Im25, with the exception of A4. Red represents positive charges and blue represents negative charges. (B) View of the UGUAA element covered with a 2Fo – Fc electron density map contoured at 0.9 σ. The UGUAA element is illustrated as a ball-and-stick scheme. (C) Structure of UGUAAA-CF Im25 complexes (PDB entry 3MDI). The RNA hexamer, UGUAAA, is bound by one molecule of CF Im25 dimer (yellow) and partially by an adjacent molecule (cyan) in the crystal. (D) View of the CF Im25-CF Im68RRM-UGUAA complex. Two RNA molecules with anti-parallel orientation bind to the same sites of the CF Im25-CF Im68RRM heterotetramer.

Full figure and legend (106K)

Yang et al. 17 reported the structure of the CF Im25-UGUAAA complex, which showed that the RNA hexamer was bound by one molecule of the CF Im25 dimer and partially by an adjacent molecule in the crystal (Figure 5C). In addition, the authors found that the CF Im25 dimer can bind two UGUA elements in solution. Consistent with this observation, our structure shows that two UGUAA elements bind simultaneously, in an anti-parallel orientation, to one CF Im25 dimer (Figure 5D). Superimposition of these two structures showed RMS deviations of Cα positions of 0.344 Å with 175 residues (Supplementary information, Figure S4C). The first three nucleotides (U1, G2 and U3) of UGUAA and UGUAAA adopt the same conformation and are recognized by CF Im25 in a similar, but not identical manner. Arg63, Phe103, Phe104, Y208 and G209 interact with the first three nucleotides of both UGUAA and UGUAAA. In the CF Im25-CF Im68RRM-UGUAA complex, UGUAA interacts with Ser58, whereas in the CF Im25-UGUAAA complex, UGUAAA interacts with Glu55, Asp57, Glu81, Thr102 and Leu106. The Glu81-U1 and Thr102-U1 interactions require a glycerol molecule. In our studies, no glycerol was added, and accordingly, no Glu81-U1 or Thr102-U1 interactions were observed. The conformation of the 3′-ends of UGUAA and UGUAAA is different. U3, A4, A5 and A6 of UGUAAA form a larger U shape, and A4 and A5 are located in the RNA-binding pocket of the adjacent dimer. In the CF Im25-UGUAAA complex, Glu55 and Thr102 interact with A4, whereas in the CF Im25-CF Im68RRM-UGUAA complex, A4 does not interact with CF Im25 and A5 interacts with Leu99 and Gly100.

CF Im25 and CF Im68RRM cooperate to bind RNA

Human PAPOLA pre-mRNA (encoding poly(A) polymerase α, PAPα) has a canonical poly(A) site and multiple copies of the UGUAA element upstream of the AAUAAA element. CF Im binds to the UGUAA elements of PAPOLA pre-mRNA and promotes 3′-end processing 7. To validate whether CF Im assembly contributes to RNA binding, two RNA segments derived from human PAPOLA pre-mRNA, comprising the UGUAA element at the 3′-end were synthesized and used in surface plasmon resonance (SPR) assays. The two RNA segments are 5′-GCUAUUUUGUAAACA-3′ (RNA1) and 5′-CUAUUUUGUAA-3′ (RNA2). CF Im25 bound to RNA1 and RNA2 with Kds of 46 and 10 μM, respectively, whereas the CF Im25-CF Im68RRM complex bound with Kds of 170 and 100 nM, respectively, (Figure 6A and 6B). Consistent with previous studies 15, CF Im68RRM alone showed almost no binding to RNA (Supplementary information, Figure S5A). These results reveal that the CF Im25-CF Im68RRM complex assembly significantly enhances RNA-binding affinity.

Figure 6.
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RNA binding analysis. Dissociation constants (Kds) were derived using the 1:1 binding model. (A) CF Im25 bound to RNA1 and RNA2 with Kds of 46 and 10 μM, respectively. (B) The CF Im25-CF Im68RRM complex bound to RNA1 and RNA2 with Kds of 170 and 100 nM, respectively. (C) Summary of the binding properties of site-directed mutants of CF Im68RRM. Mutants of CF Im68RRM were co-purified with CF Im25.

Full figure and legend (40K)

Superimposition of the CF Im25 dimer with the CF Im25-CF Im68RRM complex shows that CF Im68RRM binding does not change the overall conformation of the CF Im25 dimer, and Dettwiler et al. 15 have shown that CF Im25 binding enables CF Im68RRM to bind RNA. Thus, CF Im68RRM may directly interact with RNA to enhance RNA-binding affinity. The anti-parallel β-sheet of the RRM domain is the major RNA-binding surface, and the highly conserved aromatic and positively charged residues from RNP1 and RNP2 motifs are generally involved in interactions with consecutive RNA bases. The β-sheet of CF Im68RRM is exposed to solvent, suggesting that CF Im68RRM may bind the RNA substrate via this region. To validate this hypothesis, mutagenesis analysis was performed. Point mutations of selected residues of CF Im68RRM were generated and these mutants were co-purified with CF Im25 for RNA-binding tests. As shown in Figure 6C, the mutants, N117A, N120A and K124A, showed reduced RNA-binding affinities, whereas the mutants, Y84A, N87A and F115A, showed similar RNA-binding affinities compared with the wild-type protein. We also generated a mutant in which the aromatic residue in the RNP1 motif, F126, was replaced by an alanine, but this mutant could not be obtained as a stable protein for kinetic analysis. Y84 and F115 are located in the β-sheet, indicating that, unusually for an RRM domain, the β-sheet of CF Im68RRM is not involved in RNA recognition. N117, N120 and K124 are located in the L3 loop of CF Im68RRM, suggesting that the L3 loop of CF Im68RRM not only interacts with CF Im25 but may also interact with RNA.

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Discussion

Comparison with other RRM-protein structures

Biochemical and structural studies have revealed that RRM domains are involved in protein-protein interactions as well as in RNA recognition 23, 24. Previous studies have shown that CF Im68 interacts with CF Im25 via the RRM domain and that the substitution of two residues within the RNP2 motif (G86V, N87V) abolished interaction with CF Im25 15. We initially speculated that CF Im68RRM might interact with CF Im25 via its β-sheet. Interestingly, the structure of the CF Im25-CF Im68RRM complex shows that CF Im68RRM interacts with CF Im25 via its L1 and L3 loops, leaving the β-sheet exposed to solvent. To date, about 10 structures of RRM domain-protein/polypeptide complexes have been determined. The recognition mechanisms of proteins by RRM domains are very diverse, and no general mechanism has emerged. The RRM domain interacts with other proteins through its β-sheet 25, 26, 27, 28, or α-helices 29, 30, or α-helices and L4 loops 18, 19, 31. To date, only one RRM-protein complex structure has been reported in which the L3 loop is involved in interactions with other protein (the Mago-Y14-PYM complex 26). Comparison of the CF Im25-CF Im68RRM complex with the Mago-Y14-PYM complex shows that CF Im68RRM binds to CF Im25 in a different manner. In the Mago-Y14-PYM complex, Y14 interacts with PYM through part of its L3 loop and interacts with Mago through the entire β-sheet. In the CF Im25-CF Im68RRM complex, the β-sheet is not involved in protein recognition and the entire L3 loop is involved in RRM-protein interaction. The novel structural information presented here demonstrates the diversity of protein recognition mechanisms, which underlie RRM domain binding.

CF Im25 dimerization is crucial for UGUAA recognition and complex assembly

Recently, CF Im has been shown to be a heterotetramer in solution 17. Our structure confirms that CF Im25 and CF Im68RRM forms a heterotetramer, induced by CF Im25 dimerization. Whether this dimerization of CF Im25 is important for the activity of CF Im is of interest, and our studies indicate that this dimerization of CF Im25 is important for complex assembly. The structural and mutational data presented here show that CF Im68RRM interacts with both molecules of the CF Im25 dimer and that CF Im25 dimerization may stabilize the conformation of the L10 loop of CF Im25, which is important for CF Im68RRM binding. CF Im25 dimerization also enables the simultaneous binding of two UGUAA elements to the CF Im25 dimer. Yang et al. 17 observed that the CF Im25 dimer bound RNA containing two separated UGUAA elements with 100-fold higher affinity than RNA containing only one UGUAA element. The minimum distance between the two UGUA elements, which is required to observe this gain in affinity is five nucleotides. Unlike the structures of the UGUAAA- and UUGUAU-CF Im25 complexes, which show CF Im25 dimer binding with only one RNA molecule in the crystals 17, our structure shows that two UGUAA RNA sequences, with anti-parallel orientation, bind to one CF Im25-CF Im68RRM heterotetramer, providing structural evidence to support the simultaneous binding of two UGUAA elements to the CF Im25 dimer. The anti-parallel positioning of the two UGUAA elements and the discontiguous RNA-binding surfaces confirm the importance of the separation by a certain number of bases of the two UGUAA elements.

CF Im25 enables CF Im68 to bind RNA through stabilization of the L3 loop of CF Im68

UV-crosslinking experiments showed that CF Im68 binds to pre-mRNA substrate very weakly, but can efficiently bind to RNA upon complex formation with CF Im25 15. Our studies therefore suggest that CF Im25 may enable CF Im68 to bind RNA. Although the β-sheet of CF Im68RRM contains the conserved RNP1 and RNP2 motifs found in other RRM domains (Figure 2A) and is accessible for RNA, our studies show that it is not involved in RNA binding. Y84, N87 and F115 are located in the β1 strand, L1 loop and β2 strand, respectively. The Y84A, N87A and F115A mutants showed similar RNA-binding affinities to the wildtype, suggesting that the β-sheet does not interact with RNA. This observation is supported by the recently determined structure of the CF Im25-CF Im59RRM complex (PDB entry 3N9U). The structure of the CF Im25-CF Im59RRM complex shows that the β-sheet is buried by a helix formed by the C-terminal extension of the RRM domain (Supplementary information, Figure S6). As CF Im59 and CF Im68 have highly homologous amino acid sequences, the β-sheet of CF Im68RRM is possibly also buried by a helix and is inaccessible to RNA. Substitutions of N117, N120 and K124, which are located in the L3 loop, reduce RNA-binding affinities, suggesting the L3 loop of CF Im68RRM may bind to RNA. This observation is particularly interesting. In SXL-RRM2 20 and PABP RRM1 32, the L3 loops interact with RNA but not with protein, whereas in the Mago-Y14-PYM complex, the L3 loop interacts only with protein. To our knowledge, the RRM domain of CF Im68 is the first example of an RRM domain that binds to both protein and RNA via the L3 loop. Without CF Im25 binding, the L3 loop of CF Im68RRM is thought to be more flexible, which may affect RNA binding. Upon complex formation with CF Im25, the L3 loop of CF Im68RRM is extended and accessible for RNA binding. CF Im25 may enable CF Im68 to bind RNA in this manner.

CF Im68 makes an essential contribution to RNA binding

The CF Im25-CF Im68RRM complex binds to RNA1 and RNA2 much more efficiently than CF Im25 alone (270-fold for RNA1 and 100-fold for RNA2), indicating that the large subunit, CF Im68, makes an essential contribution to pre-mRNA recognition. Point mutation and kinetics analyses show that the L3 loop of CF Im68RRM may be involved in RNA binding. As the UGUAA element orientates opposite to CF Im68RRM from the 5′ to 3′-end, the L3 loop of CF Im68RRM may bind the immediately flanking upstream region of the UGUAA element. Complex assembly places the L3 loop of CF Im68RRM and the NUDIX domain of CF Im25 together, forming a large and continuous RNA-binding platform (Supplementary information, Figure S7A). We observed that CF Im25 binds to RNA1 much more weakly than RNA2 (about fivefold). The program UNAFold 33 indicated that RNA1 folds into a hairpin structure (Supplementary information, Figure S7B). The U-A and G-C intramolecular interactions bury the UGUAA element, impairing the binding of CF Im25. Pre-mRNA processing, such as splicing, is influenced by the secondary structure of the pre-mRNA 34. Most proteins involved in splicing regulation recognize single-stranded, rather than base-paired RNA. The hairpin structure of RNA reduces the binding of CF Im25 by about fivefold but has a much smaller impact on the CF Im25-CF Im68RRM complex (less than twofold), suggesting that CF Im68 binding helps CF Im25 to recognize the UGUAA element, besides promoting a higher binding affinity.

During 3′-end processing, CF Im binds to pre-mRNA concomitantly with CPSF to stabilize the binding of CPSF to the AAUAAA hexamer at an early stage of processing complex assembly 5, 6. It binds to the UGUAN element upstream of the poly(A) site in a sequence-dependent manner 4, 35. The CF Im25 dimer simultaneously binds to two UGUA elements within one molecule of pre-mRNA (termed as two UGUAA elements binding mode) 17. In addition, CF Im25 and CF Im68 cooperates to bind the UGUAA element and the immediately upstream flanking region, respectively, for higher affinity (termed as synergistic binding mode). Bioinformatic analyses revealed that the two modes are both functionally important for pre-mRNA recognition by CF Im (Table 2). Approximately 43.6% of human mRNAs (a total number of 44 563) contain A(A/U)UAAA elements immediately upstream of the poly(A) site and UGUAN elements upstream of A(A/U)UAAA and 29.3% contain multiple copies of UGUAN elements. We also analyzed the mRNAs from Mus musculus, Danio rerio, Gallus gallus and Xenopus laevis and found that the proportions are 42.8% and 28%, 38.6% and 28.9%, 27.8% and 19.7%, and 65.9% and 50.7%, respectively (Table 2). These results show that CF Im binds to about half of all pre-mRNAs, of which the majority contain at least two copies of UGUAN elements and the minority contain only one copy of the UGUAN element. CF Im may bind to pre-mRNAs containing at least two copies of UGUAN elements via both the two UGUA element-binding and the synergistic-binding modes. For the pre-mRNAs containing only one copy of the UGUAN element, which is the case for about 10% of all mRNAs, CF Im binds the pre-mRNA via the synergistic binding mode to ensure efficient binding.


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Materials and Methods

Protein expression and purification

The cDNAs of CF Im25 (residues 34-227) and CF Im68RRM (residues 78-159) were cloned into the vector pET22b. Cys159 of CF Im68RRM was replaced by an alanine to prevent disulfide bond formation. GST or GST proteins were cloned into the pGEX-4T-2 vector. His-MBP-tag proteins were cloned into the modified pET32a vector, the products of which contain His-MBP-tags in the N-terminal of the recombinant proteins. Proteins were expressed in Escherichia coli Rosetta (DE3) (Novagen). Mutations were introduced using PCR by designing mutated residues in primers with the MutanBEST kit (TAKARA). All plasmids were confirmed by DNA sequencing. CF Im25 and CF Im68RRM were co-purified by Ni2+ ion-affinity chromatography, and further purified by Superdex-200 gel filtration and monoQ ion exchange chromatography (GE Healthcare). Finally, the CF Im68-CF Im68RRM complex was concentrated by centrifugal ultrafiltration (Millipore, 5 kDa cutoff) to approximately 8 mg/ml, as estimated using the BCA Protein Assay Kit (Pierce), in 2 mM Tris (pH 8.0), and 100 mM NaCl for crystallization assays.

Crystallization and data collection

The CF Im25-CF Im68RRM complex was crystallized at the concentration of approximately 8 mg/ml by hanging-drop vapor diffusion at 10 °C with the reservoir solution containing 16% PEG3350, 5% dioxane and 0.1 M sodium citrate, pH 5.0. The 2.7 Å diffraction data was collected at beamline 3W1A of Beijing Synchrotron Radiation Facility (BSRF). The CF Im25-CF Im68RRM complex was incubated with twofold excess of UGUAA (TAKARA) on ice for 0.5 h, with a final concentration of approximately 6 mg/ml. The CF Im25-CF Im68RRM-UGUAA complex was crystallized by sitting-drop vapor diffusion at 10 °C with the reservoir solution containing 15% PEG3350, 9% dioxane and 0.1 M sodium citrate, pH 5.0. The 2.9 Å diffraction data was collected at beamline BL17U of Shanghai Synchrotron Radiation Facility (SSRF). The data were processed and scaled with the HKL2000 package 36.

Structure determination

The CF Im25-CF Im68RRM crystals belong to space group C2 with the cell parameters: a = 159.77 Å, b = 105.51 Å, c = 146.53 Å, and α = γ = 90.00°, β = 112.58°. Each asymmetric unit contains three copies of a 2:2 CF Im25-CF Im68RRM complex. The phase was determined by molecular replacement using Molrep 37 and Phaser 38. First, six CF Im25 subunits were found using the structure of apo CF Im25 (PDB entry 2cl3) as the search model and then fixed. Then six CF Im68RRM subunits were found using the structure of RBMY protein RRM domain (PDB entry 2FY1) as the search model. The model completeness was carried out in COOT 39 and the refinement was performed by REFMAC5 40 with non-crystallographic symmetry (NCS) restraints and CNS 41. The NCS restraints were tight in earlier stages and completely relaxed in later stages. Water molecules were added to the model by inspection of 2Fo – Fc and Fo – Fc density maps in the final stages. The final refined model has an R factor (Rfree) of 21.4% (26.5%). The quality of model was checked using the program Molprobity 42. The Ramachandran plot reveals that 95.5% of the residues are in the most favored region, with an additional 4.4% in the additionally allowed region. One residue is in the outlier region, which is Pro113. The CF Im25-CF Im68RRM-RNA crystals belong to space group P21 with the cell parameters: a = 104.78 Å, b = 129.42 Å, c =111.16 Å, and α = γ = 90.00°, β = 94.01° . Each asymmetric unit contains four copies of CF Im25-CF Im68RRM heterotetramer. The phase was determined by molecular replacement with Phaser 38. Seven RNA molecules can be traced in the electron density map within the cavities of the CF Im25 subunits, whereas no electron density for RNA was observed within the same region of the remaining CF Im25 subunit. Thus, seven RNA chains (termed chain Q, R, S T, U, V and W, respectively) were manually added with the guidance of 2Fo – Fc and Fo – Fc density maps in Coot 39. Chain Q contains U1 and the guanine base of G2, chain R contains all the five nucleotides with the exception of the adenine base of A4 and the phosphate group of A5, chain S contains all the five nucleotides, chain T contains the first three nucleotides and A5 with the exception of the phosphate group of A5, chain U contains the first three nucleotides and the adenine base of A5, chain V contains the first three nucleotides and chain W contains U1. The refinement was performed by REFMAC5 40. Finally, water molecules were added to the model by inspection of 2Fo – Fc and Fo – Fc density maps. The translation-libration-screw (TLS) model was applied near the end of refinement. The final refined model has an R factor (Rfree) of 22.6% (27.6%) and was validated using Molprobity 42. The Ramachandran plot revealed that 97.3% of the residues are in the most favored region, with an additional 2.7% in the additionally allowed region. Structural figures were prepared with Pymol (http://www.pymol.org).

Glutathione S-transferase pull-down assays

Small-scale pull-down assays were performed. GST-proteins or GST tag (150 μg) from the soluble fraction of E. coli cell lysate was incubated with 75 μl of glutathione agarose beads (GE Healthcare) for 30 min at room temperature. After washing three times with binding buffer, beads were incubated with 150 μg of purified recombinant proteins for 1 h at 16 °C. After incubation, the beads were washed three times with binding buffer to remove unbound proteins. Bound proteins were eluted from the beads by boiling in SDS sample buffer and resolved on SDS-PAGE.

Immunoblot analysis

An aliquot containing 50 μg of protein from the soluble fraction of E. coli cell lysate expressing GST or GST-proteins was incubated with 20 μl of glutathione agarose beads (GE Healthcare) for 20 min at room temperature. After washing four times with 1× PBS, beads bound with GST or GST-proteins were incubated with 50 μg of the recombinant His-MBP-tagged or His-tagged proteins in 0.25 ml HNTG-buffer (20 mM Hepes-KOH, pH 7.5, 100 mM NaCl, 0.1% Triton X-100, and 10% glycerol) for 1 h at 4 °C. After incubation, the beads were washed six times with 1 ml HNTG buffer to remove unbound proteins. Bound proteins were eluted from the beads by boiling in SDS sample buffer and detected by immunoblot analysis.

Size-exclusion chromatography assay

The size-exclusion chromatography assays were performed with a Superdex 200 column (10/300 GL) (GE Healthcare). The protein sample or molecular mass standards were applied to the Superdex 200 column (10/300 GL) and eluted with 50 mM Tris, pH 8.0 and 200 mM NaCl. Standard proteins (GE Healthcare) were thyroglobulin (669.0 kDa), ferritin (440.0 kDa), albumin (69.0 kDa), ovalbumin (43.0 kDa), ribonuclease A (13.7 kDa). The void volume was determined with blue dextran (GE Healthcare).

Surface plasmon resonance measurement

All SPR studies were performed with a Biacore 3000 instrument (Biacore AB, Uppsala, Sweden). The RNA segment with the same sequence found upstream of the human PAPOLA pre-mRNA poly(A) site (5′-GCUAUUUUGUAAACA-3′ (−63 to −49) or 5′-CUAUUUUGUAA-3′ (−62 to −42)) was immobilized on a SA chip via a biotin at the 3′-end, and solutions containing wild-type proteins and mutants at different concentrations were passed over the chip at 10 μl/min and washed by 50 mM NaOH. All experiments were carried out at 25 °C in binding buffer (20 mM Tris, pH 8.0, 150 mM NaCl). The binding curves were fitted according to a one-site binding model using Origin software (http://www.originlab.com). The raw sensorgrams data obtained with different concentrations of proteins are provided as Supplementary information, Figure S5.

Accession codes

The coordinates and structure factors have been deposited with accession number 3P5T (for the CF Im25complex-CF Im68RRM complex) and 3P6Y (for the CF Im25-CF Im68RRM-UGUAA complex).

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Acknowledgements

We thank the staff at BSRF beamline 3W1A and SSRF beamline BL17U for assistance with synchrotron data collection and Dr S Barabino (University of Milano-Bicocca) for plasmids, and Dr C Zhao and Dr J Zang (University of Science and Technology of China) for their valuable advice on experiments and manuscript. Financial support for this project was provided by research grants from the National Natural Science Foundation of China (30025012, 30900224 and 10979039), the Chinese Ministry of Science and Technology (2006CB806500, 2006CB910200 and 2006AA02A318), the Chinese Academy of Sciences (KSCX2-YW-R-60), the Chinese Ministry of Education (20070358025) and the Natural Science Foundation of Anhui Province (090413081). Heng Li was supported by the Graduate Innovation Foundation of USTC (KD2007034).

(Supplementary information is linked to the online version of the paper on the Cell Research website.)