Architecture and nucleic acids recognition mechanism of the THO complex, an mRNP assembly factor
2012; Springer Nature; Volume: 31; Issue: 6 Linguagem: Inglês
10.1038/emboj.2012.10
ISSN1460-2075
AutoresÁlvaro Peña, Kamil Gewartowski, Seweryn Mroczek, Jorge Cuéllar, A. Szykowska, Andrzej Prokop, Mariusz Czarnocki‐Cieciura, Jan Piwowarski, Cristina Tous, Andrés Aguilera, José L. Carrascosa, José Valpuesta, Andrzej Dziembowski,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle7 February 2012free access Architecture and nucleic acids recognition mechanism of the THO complex, an mRNP assembly factor Álvaro Peña Álvaro Peña Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), Department of Molecular Biology, CSIC, Madrid, Spain Search for more papers by this author Kamil Gewartowski Kamil Gewartowski Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Seweryn Mroczek Seweryn Mroczek Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Jorge Cuéllar Jorge Cuéllar Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), Department of Molecular Biology, CSIC, Madrid, Spain Search for more papers by this author Aleksandra Szykowska Aleksandra Szykowska Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Andrzej Prokop Andrzej Prokop Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Mariusz Czarnocki-Cieciura Mariusz Czarnocki-Cieciura Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Jan Piwowarski Jan Piwowarski Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Cristina Tous Cristina Tous Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla–CSIC, Sevilla, Spain Search for more papers by this author Andrés Aguilera Andrés Aguilera Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla–CSIC, Sevilla, Spain Search for more papers by this author José L Carrascosa José L Carrascosa Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), Department of Molecular Biology, CSIC, Madrid, Spain Search for more papers by this author José María Valpuesta Corresponding Author José María Valpuesta Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), Department of Molecular Biology, CSIC, Madrid, Spain Search for more papers by this author Andrzej Dziembowski Corresponding Author Andrzej Dziembowski Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Álvaro Peña Álvaro Peña Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), Department of Molecular Biology, CSIC, Madrid, Spain Search for more papers by this author Kamil Gewartowski Kamil Gewartowski Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Seweryn Mroczek Seweryn Mroczek Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Jorge Cuéllar Jorge Cuéllar Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), Department of Molecular Biology, CSIC, Madrid, Spain Search for more papers by this author Aleksandra Szykowska Aleksandra Szykowska Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Andrzej Prokop Andrzej Prokop Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Mariusz Czarnocki-Cieciura Mariusz Czarnocki-Cieciura Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Jan Piwowarski Jan Piwowarski Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Cristina Tous Cristina Tous Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla–CSIC, Sevilla, Spain Search for more papers by this author Andrés Aguilera Andrés Aguilera Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla–CSIC, Sevilla, Spain Search for more papers by this author José L Carrascosa José L Carrascosa Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), Department of Molecular Biology, CSIC, Madrid, Spain Search for more papers by this author José María Valpuesta Corresponding Author José María Valpuesta Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), Department of Molecular Biology, CSIC, Madrid, Spain Search for more papers by this author Andrzej Dziembowski Corresponding Author Andrzej Dziembowski Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland Search for more papers by this author Author Information Álvaro Peña1,‡, Kamil Gewartowski2,3,‡, Seweryn Mroczek2,3, Jorge Cuéllar1, Aleksandra Szykowska2,3, Andrzej Prokop2,3, Mariusz Czarnocki-Cieciura2,3, Jan Piwowarski3, Cristina Tous4, Andrés Aguilera4, José L Carrascosa1, José María Valpuesta 1 and Andrzej Dziembowski 2,3 1Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), Department of Molecular Biology, CSIC, Madrid, Spain 2Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland 3Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland 4Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla–CSIC, Sevilla, Spain ‡These authors contributed equally to this work *Corresponding authors: Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), CSIC, Madrid 28049, Spain. Tel: +34 915854690, Fax: +34 915854506: E-mail: [email protected] of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw 02-106, Poland. Tel: +48 225922033; Fax: +48 8237189; E-mail: [email protected] The EMBO Journal (2012)31:1605-1616https://doi.org/10.1038/emboj.2012.10 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The THO complex is a key factor in co-transcriptional formation of export-competent messenger ribonucleoprotein particles, yet its structure and mechanism of chromatin recruitment remain unknown. In yeast, this complex has been described as a heterotetramer (Tho2, Hpr1, Mft1, and Thp2) that interacts with Tex1 and mRNA export factors Sub2 and Yra1 to form the TRanscription EXport (TREX) complex. In this study, we purified yeast THO and found Tex1 to be part of its core. We determined the three-dimensional structures of five-subunit THO complex by electron microscopy and located the positions of Tex1, Hpr1, and Tho2 C-terminus using various labelling techniques. In the case of Tex1, a β-propeller protein, we have generated an atomic model which docks into the corresponding part of the THO complex envelope. Furthermore, we show that THO directly interacts with nucleic acids through the unfolded C-terminal region of Tho2, whose removal reduces THO recruitment to active chromatin leading to mRNA biogenesis defects. In summary, this study describes the THO architecture, the structural basis for its chromatin targeting, and highlights the importance of unfolded regions of eukaryotic proteins. Introduction mRNA biogenesis and export is a very complex process involving transient interactions between a large number of proteins and assemblies. During transcription elongation, pre-mRNA molecules are packed into RNA–protein assemblies termed mRNPs (Kohler and Hurt, 2007). All steps leading to the production of translation-competent mRNA in the cytoplasm (transcription, mRNA processing, and export from the nucleus) are tightly coupled, and impairment of any step leads to the activation of the RNA surveillance pathway and the consequent degradation of improper mRNA molecules (Houseley et al, 2006). THO is an evolutionarily conserved macromolecular assembly that functions during transcription facilitating the mRNP packaging and export. The yeast THO complex associates with chromatin in a transcription-dependent manner and is essential for efficient co-transcriptional recruitment of mRNA export factors Yra1 and Sub2 (Strasser et al, 2002). Therefore, THO plays an important role in coupling transcription to mRNA export, although its precise function is still elusive. Yeast THO has been described as a four-subunit complex composed of Tho2 (180 kDa), Hpr1 (90 kDa), Mft1 (45 kDa), and Thp2 (30 kDa) (Chavez et al, 2000), for which no structural information is available. It has been shown that THO interacts with three other proteins, the RNA helicase Sub2, the RNA-binding protein Yra1, and Tex1 whose function is unknown, forming the TREX complex—from TRanscription and Export (Strasser et al, 2002). Moreover, during transcription, THO interacts with mRNA export factor Mex67, and the serine–arginine-rich (SR)-like proteins Gbp2 and Hrb1 (Strasser et al, 2002; Zenklusen et al, 2002; Hurt et al, 2004). It has also recently been published that TREX interacts with the Prp19 complex, involved in splicing and transcription elongation (Chanarat et al, 2011). Inactivation of the THO subunits results in remarkable molecular phenotypes, which reveals the important role of the complex in mRNA biogenesis and genome stability. Lack of THO causes impairment of mRNP formation leading to defects in transcription elongation and to the formation of RNA/DNA hybrids (R-loops), which in turn causes genomic instability (Huertas and Aguilera, 2003). Another interesting phenotype generated by THO deletion is the formation of large aggregates (called heavy chromatin) composed of transcriptionally active chromatin, nascent transcripts, RNA export machinery, and nuclear pore complexes (NPC) (Rougemaille et al, 2008). Finally, the expression level of long GC-rich genes (like LacZ) is markedly reduced in tho mutants (Chavez et al, 2001). All these phenotypes together with the interaction of THO with export factors described above strongly suggest that its activity is directly associated with mRNA transcription, biogenesis, and export. The interaction between THO and active chromatin, together with biochemical studies of Yra1 and Sub2, have led to a model in which the THO/TREX complex is recruited to mRNA at the early stages of the export pathway (Strasser et al, 2002; Zenklusen et al, 2002). Then, mRNA is transmitted to the Mex67/Mtr2 export receptor, which interacts with phenylalanine-glycine (FG) repeat-containing nuclear pore proteins, thus facilitating mRNA translocation through the NPC (Reed and Cheng, 2005; Kohler and Hurt, 2007). Despite numerous studies, the mechanism of THO function is not well defined. So far, no structural information regarding THO and its subunits has been published and it is unclear whether it interacts directly with nucleic acids and how it is recruited to chromatin. Interestingly, however, a recent report suggests that THO interaction with active chromatin is partially dependent of Syf1—a subunit of the Prp19 complex involved in transcription elongation and splicing (Chanarat et al, 2011). In this work we provide mechanistic insight into the THO function. We demonstrate that Tex1 interacts stably with THO as it co-purifies with the other subunits even at high salt concentrations. We present the three-dimensional reconstruction of the five subunits of the THO complex and the localization, within the structure, of the subunits Tho2, Hpr1, and Tex1. Furthermore, we show that the largest THO subunit Tho2, and in particular its C-terminal domain, is directly responsible for interaction with nucleic acids (ssDNA, dsDNA, and RNA). Deletion of this fragment, while not altering the assembly of the complex, leads to defects in mRNA biogenesis and increases genome instability. Most importantly, the intrinsically unfolded C-terminus of Tho2 is essential for efficient recruitment of the THO complex to chromatin. Results Purification and structural characterization of the THO complex To characterize the THO complex, we devised an efficient purification procedure. The native THO complex was purified by IgG affinity chromatography followed by ion exchange chromatography using a Saccharomyces cerevisiae strain with a TAP-tagged Tho2 protein (Dziembowski and Seraphin, 2008). The purified complex reproducibly contained not only the four-core THO subunits (Tho2, Hpr1, Mtf2, and Thp2) but also the TREX component Tex1 (Figure 1A; Supplementary Figure S1), while the other TREX components (Yra1 and Sub2) were conspicuously absent. In the present manuscript, we refer to the five-subunit assembly as the THO complex (Jimeno et al, 2002; Strasser et al, 2002; Rehwinkel et al, 2004). The purified THO complex isolated from S. cerevisiae was not very soluble (<1 mg/ml), too low to attempt crystallization, but sufficient for structural analysis by electron microscopy. Aliquots of THO were negatively stained and observed by electron microscopy (Figure 1B), which revealed the presence of a homogeneous population of long, thin particles (top gallery in Figure 1B). A total of 14 115 particles were selected, aligned, and classified as described in Materials and methods. A maximum-likelihood classification revealed as the largest population a croissant-like structure ∼220 Å long and ∼115 Å high, with a flat surface at the base and two large protrusions at the top, one long and thin and the other shorter but wider (Figure 1C). From the tip of the larger protrusion stems a thin and flexible stain-excluding mass (arrow in Figure 1C), which appears to be sticky as suggested by the presence (∼20% of the population) of dimers of THO complexes interacting through this region and forming butterfly like structures (see the bottom gallery in Figure 1B and the average image of 1800 particles in Figure 1D). Figure 1.Three-dimensional reconstruction of the THO complex. (A) SDS–PAGE of the THO complex purified by affinity chromatography followed by ion exchange chromatography, which reproducibly showed the presence in stoichiometric amounts of the four canonical THO subunits (Tho2, Hpr1, Mft1, Thp2) and Tex1. (B) An electron microscopy negatively stained field of THO particles. Bar indicates 1000 Å. The top gallery shows a selection of THO particles and the bottom gallery, a selection of double, butterfly like THO particles. (C) Two-dimensional average image of the THO complex. Arrow points to the flexible region described in the text. (D) Two-dimensional average image of the double, THO particle. Bar indicates 100 Å in (C, D). (E) Four orthogonal views of the three-dimensional reconstruction of the THO complex. Bar indicates 100 Å. Download figure Download PowerPoint The three-dimensional reconstruction generated using these particles (∼17 Å resolution) revealed in full detail the features described above (Figure 1E; Supplementary Figures S2–S5). It is important to point out that the flexible mass stemming from the long protrusion was not observed in the reconstruction, probably due to an averaging out caused by the presence of different conformations of this domain. Mapping of Tex1 and Hpr1 into the THO complex To further characterize the structure of the THO complex, we sought to locate some of the subunits within the complex. We first assessed the position of Tex1. For this purpose, a new strain lacking Tex1 (Tho2TAPΔTex1) was constructed with a TAP-tagged Tho2 subunit. A homogeneous, stoichiometric complex composed of Tho2, Hpr1, Mtf2, and Thp2 proteins was purified using the protocol described above (Figure 2A). Aliquots of the complex were subsequently negatively stained and observed by electron microscopy (Figure 2B), which again revealed the presence of a homogeneous population of long, thin particles. The two-dimensional average image obtained after two-dimensional maximum-likelihood classification and averaging of the largest population selected from 13 273 particles (inset in Figure 2B) revealed a similar overall structure to that obtained for the THO complex but lacking one of the protruding masses. This was confirmed by the three-dimensional reconstruction (∼20 Å resolution), which showed the same long, thin, and asymmetric structure, albeit lacking the wider protruding mass in the centre of the THO structure (Figure 2C). Therefore, we assign this protrusion to Tex1. Figure 2.Three-dimensional reconstruction of the THOΔTex1 complex and mapping of Tex1 and Hpr1 into the THO complex. (A) SDS–PAGE of THO complexes purified by affinity chromatography from a Tho2TAP and Tho2TAPΔTex1 S. cerevisiae strains. The right lane shows that THO complex can assemble without Tex1 protein. (B) An electron microscopy field of negatively stained THOΔTex1 particles. The inset shows the two-dimensional average image of the most common view of the complex. Bar indicates 1000 Å in the micrograph and 100 Å in the inset. (C) Four orthogonal views of the three-dimensional reconstruction of THOΔTex1. Bar indicates 100 Å. (D) Atomic model of the N-terminal, β-propeller domain of Tex1. (E) Docking of the atomic model of Tex1 into the corresponding mass of the THO complex. (F) The same docking in an orthogonal, cut section of the complex. The arrow points to the putative region filled by the non-reconstructed, C-terminal region of Tex1. (G) Two-dimensional averages of the THO complex (left) and the immunocomplex formed between THO and the anti-Hpr1 polyclonal antibody (right). Download figure Download PowerPoint Tex1 has been described to contain several WD40 domains, which could give rise to a β-propeller structure (Rehwinkel et al, 2004). We generated an atomic model of residues 47–371 of Tex1 (residues 1–46 and 372–422 could not be modelled into any known structure), which revealed a seven-blade β-propeller structure (Figure 2D). The atomic model was subsequently docked into the three-dimensional reconstruction of the THO complex. Docking, either manual or automatic, suggested that the atomic model of Tex1 fitted well into the part of the THO volume in which Tex1 was mapped to (Figure 2E and F), leaving only a small region that probably contains the non-modelled C-terminal region of the protein (arrow in Figure 2F). To locate other subunits of the THO complex we performed immunomicroscopy, using either specific antibodies against the different subunits or epitope-tagged subunits. These approaches produced inconclusive results, with the exception of one polyclonal antibody against the C-terminal region of Hpr1, which stably associated with the THO complex. Aliquots of the immunocomplex were negatively stained and a total of 8250 particles were selected and processed. The two-dimensional average obtained revealed an extra mass (arrow in Figure 2G) that corresponds to the Fab domain of the antibody bound to the wider end of the THO structure (Figure 2G), indicating the position of the Hpr1 subunit. The C-terminal region of the Tho2 protein interacts with nucleic acids THO has been shown to associate with RNA and DNA in vitro (Jimeno et al, 2002), but its function and the subunit(s) involved in this interaction are not known. To characterize these interactions, we performed UV cross-linking experiments using the highly purified complex and 32P-labelled oligonucleotides (either RNA, ssDNA, or dsDNA) (Figure 3A). The three types of oligonucleotides were cross-linked to the largest subunit of the THO complex, Tho2. These interactions were also confirmed by an electrophoretic mobility shift assay (data not shown). Figure 3.The C-terminal region of Tho2 interacts with nucleic acids. (A) Tho2 cross-links to RNA, ssDNA, or dsDNA. Purified THO was incubated with radiolabelled oligonucleotides (either RNA, ssDNA, or dsDNA) and UV cross-linked separated by SDS–PAGE. Proteins were visualized by Coomassie blue staining (left) while cross-linked radiolabelled nucleic acid was visualized using autoradiography (right). (B) Diagram of a modified Tho2 protein showing the position of the C3 protease site and the TAP-tag. (C) The C-terminal region of Tho2 is essential for Tho2–RNA interaction. A C3 cleavage site was introduced in Tho2 after residues 567aa (Tho2 567-C3 TAP) and 1270 (Tho2 1270-C3 TAP), and the complex subsequently treated with C3 protease and purified by ion exchange chromatography. Afterwards, the complex was UV cross-linked to 32P-labelled in vitro transcribed RNA. Products of cross-linking reactions were treated with RNase A and separated by SDS–PAGE. Proteins were visualized by Coomassie blue staining (lanes a) while cross-linked radiolabelled nucleic acid was visualized using autoradiography (lanes b). The Tho2 proteolysis products and visible Tho2 cross-links are indicated. (D) The C-terminal region of Tho2 cross-links to RNA. The THO complex with the Tho2 1270-C3 TAP mutant was C3 protease digested and processed as described above. Novel cross-linking product with molecular weight corresponding to the small C-terminal fragment of the Tho2 protein is indicated. Download figure Download PowerPoint The analysis of the Tho2 sequence did not reveal any canonical RNA-binding domains and secondary structure predictions analyses suggested that it is composed mostly of α-helices. Therefore, to identify which part of the very large Tho2 polypeptide interacts with nucleic acids, we performed cross-linking experiments combined with site-specific protease digestion of this protein. For this purpose, we introduced C3 protease cleavage sites at positions 567 (Tho2 567-C3 TAP) or 1270 (Tho2 1270-C3 TAP) of the Tho2 polypeptide in the Tho2TAP strain (see diagram in Figure 3B). The modified THO complexes were purified by IgG affinity chromatography followed by C3 protease cleavage and ion exchange chromatography and then subjected to UV cross-linking (Figure 3C). In the case of Tho2 567-C3 TAP, only a small percentage of Tho2 was proteolyzed, and the RNA remained associated with the intact Tho2 and to the C-terminal fragment excised (Figure 3C, lanes 4a and 4b), which points to this region as involved in RNA binding. In contrast, the digestion of Tho2 1270-C3 TAP was complete, but only the large N-terminal fragment remained associated with the THO complex while the C-terminal 327 aa fragment was lost (Figure 3C, lane 6). Interestingly, RNA-Tho2 cross-link disappeared when the C-terminal fragment was removed, which reinforces the notion of this part of the Tho2 protein being involved in the interaction with RNA. To confirm that the C-terminal region of the Tho2 indeed interacts with RNA, we altered the order of the procedure and performed protease digestion after all the chromatography steps (Figure 3D). This ensures that both fragments appearing after proteolysis are present in the cross-linking solution. After UV exposure in case of Tho2 1270-C3 TAP, a new cross-linking species was visible at a molecular weight corresponding to the small C-terminal fragment of the Tho2 protein. We conclude that the C-terminal fragment of Tho2 interacts with nucleic acids. The C-terminal region of Tho2 constitutes a basic unfolded tail not essential for complex integrity We set out to characterize the C-terminal, nucleic acid binding region (residues 1279–1597) of Tho2. A bioinformatics analysis of this region using several Tho2 sequences revealed a poorly conserved, highly positively charged and partly disordered region (see the multiple alignment of Tho2 sequences; Supplementary Figure S6A). This region was insoluble when expressed in Escherichia coli, so in order to locate the secondary structure elements and the unstructured regions, we combined trypsin digestion and CD analysis of the fragments. We generated 44 constructs encompassing different fragments of the nucleic acid binding region, which exhibited different degrees of solubility, but most of which were highly sensitive to trypsin digestion. The 1411–1530 region was exceptionally sensitive, containing no stable fragments at all (Figure 4A) and its CD spectrum showed minimal values for wavelengths below 200 nm (Figure 4B), suggesting a high level of disorder. In addition, we acquired CD spectra at higher temperatures to see if there are any secondary structures to be destabilized by heat, but did not detect any significant differences (Supplementary Figure S6B). In contrast, the CD spectrum of the 1279–1433 fragment showed a high content of α-helices (Figure 4B) and limited proteolysis (Figure 4A) combined with mass spectrometry indicated that it forms a stable fragment between residues 1279–1405. This region, when expressed in E. coli, was highly soluble and folded correctly, as expected (Figure 4B). According to the CD spectra of both the 1279–1433 and 1279–1405 fragments, their secondary structures were stable up to 45°C and completely melted at 60°C (Supplementary Figure S6B). Therefore, we suggest that the disordered domain in the C-terminal region of Tho2 is located at the very end of the polypeptide chain (residues 1405–1597). Figure 4.The C-terminal region of Tho2 forms a basic unfolded tail essential for THO complex nucleic acids binding. (A) Limited proteolysis experiments of the recombinant Tho2 protein fragments. (B) Circular dichroism spectra of the recombinant Tho2 protein fragments. Notice that Tho21411−1530 fragment generates a minimum below 200 nm characteristic of disordered proteins, while Tho21279−1404 and Tho21279−1433 have minima around 210 and 230, suggestive of a high α-helix content. (C) UV cross-linking between nucleic acids and THO complexes isolated from Tho2TAP; Tho2Δ1271–1597 and Tho2Δ1408–1597 strains. Purified THO complexes were incubated with radiolabelled RNA, ssDNA, or dsDNA. After cross-linking, the proteins were separated by SDS–PAGE. Proteins were visualized by Coomassie blue staining (left) while cross-linked radiolabelled nucleic acid was visualized using autoradiography (right). Fast migrating radioactive species (marked with asterisks) represent unbound dsDNA particles. Download figure Download PowerPoint Taken together, the results presented above strongly suggest that the region of Tho2 responsible for the interaction with nucleic acids is partially disordered. Although the amino-acid sequence of this fragment is not evolutionary conserved, all the Tho2 sequences analysed contain a large number of basic residues, suggesting that its function may be preserved in other eukaryotes. To further analyse the role of the Tho2 C-terminal region, we constructed yeast strains expressing two shortened, TAP-tagged versions of Tho2: Tho2Δ1271–1597 TAP, where the entire region was removed, and Tho2Δ1408–1597 TAP, where only the unstructured part was deleted. When purified by ion exchange chromatography, the THO complexes with deletion mutants of Tho2 eluted from the ion exchange column at the same salt concentration as the full-length version (Supplementary Figure S1). Also, the composition of the complexes was unaltered as revealed by SDS–PAGE. These results strongly indicated that the C-terminal region of Tho2 is dispensable for complex assembly. However, the shortened versions of Tho2 were virtually unable to bind any types of nucleic acids (Figure 4C). In contrast, Tex1 appeared not to be involved in THO interaction with nucleic acids, as there was no difference between cross-linking of nucleic acids using the standard THO and the THOΔTex1 complex (Supplementary Figure S6C). The C-terminal region of the Tho2 protein is located at the tip of the narrow protrusion within the THO complex structure We then set out to locate the C-terminal region of Tho2 in the THO complex by performing three-dimensional reconstructions of the two complexes containing Tho2Δ1271–1597 and Tho2Δ1408–1597. We expected to see a volume missing from the original structure of the complex; however, we observed no significant differen
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