The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform
Alfredo Castello - One of the best experts on this subject based on the ideXlab platform.
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Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap
Nature Protocols, 2017Co-Authors: Alfredo Castello, Aino I. Järvelin, Bernd Fischer, Christian K. Frese, Rastislav Horos, Anne-marie Alleaume, Sophia Foehr, Tomaz Curk, Jeroen Krijgsveld, Matthias W. HentzeAbstract:This protocol is an extension to: Nat. Protoc. 8 , 491–500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013 RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native Interactions with RNA. In brief, cells are irradiated with UV light to induce protein–RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein–RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supeRNAtant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown. Here the authors provide an extension to their earlier RNA interactome capture protocol. This Protocol Extension describes RBDmap—a method to identify the regions of RNA-binding proteins engaged in native Interactions with RNA, in a proteome-wide manner.
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Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap
Nature protocols, 2017Co-Authors: Alfredo Castello, Aino I. Järvelin, Bernd Fischer, Christian K. Frese, Rastislav Horos, Anne-marie Alleaume, Sophia Foehr, Tomaz Curk, Jeroen Krijgsveld, Matthias W. HentzeAbstract:This protocol is an extension to: Nat. Protoc. 8, 491-500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native Interactions with RNA. In brief, cells are irradiated with UV light to induce protein-RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein-RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supeRNAtant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown.
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The new (dis)order in RNA regulation
Cell Communication and Signaling, 2016Co-Authors: Aino I. Järvelin, Marko Noerenberg, Ilan Davis, Alfredo CastelloAbstract:RNA-binding proteins play a key role in the regulation of all aspects of RNA metabolism, from the synthesis of RNA to its decay. Protein-RNA Interactions have been thought to be mostly mediated by canonical RNA-binding domains that form stable secondary and tertiary structures. However, a number of pioneering studies over the past decades, together with recent proteome-wide data, have challenged this view, revealing surprising roles for intrinsically disordered protein regions in RNA binding. Here, we discuss how disordered protein regions can mediate protein-RNA Interactions, conceptually grouping these regions into RS-rich, RG-rich, and other basic sequences, that can mediate both specific and non-specific Interactions with RNA. Disordered regions can also influence RNA metabolism through protein aggregation and hydrogel formation. Importantly, protein-RNA Interactions mediated by disordered regions can influence nearly all aspects of co- and post-transcriptional RNA processes and, consequently, their disruption can cause disease. Despite growing interest in disordered protein regions and their roles in RNA biology, their mechanisms of binding, regulation, and physiological consequences remain poorly understood. In the coming years, the study of these unorthodox Interactions will yield important insights into RNA regulation in cellular homeostasis and disease.
Matthias W. Hentze - One of the best experts on this subject based on the ideXlab platform.
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Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap
Nature Protocols, 2017Co-Authors: Alfredo Castello, Aino I. Järvelin, Bernd Fischer, Christian K. Frese, Rastislav Horos, Anne-marie Alleaume, Sophia Foehr, Tomaz Curk, Jeroen Krijgsveld, Matthias W. HentzeAbstract:This protocol is an extension to: Nat. Protoc. 8 , 491–500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013 RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native Interactions with RNA. In brief, cells are irradiated with UV light to induce protein–RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein–RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supeRNAtant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown. Here the authors provide an extension to their earlier RNA interactome capture protocol. This Protocol Extension describes RBDmap—a method to identify the regions of RNA-binding proteins engaged in native Interactions with RNA, in a proteome-wide manner.
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Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap
Nature protocols, 2017Co-Authors: Alfredo Castello, Aino I. Järvelin, Bernd Fischer, Christian K. Frese, Rastislav Horos, Anne-marie Alleaume, Sophia Foehr, Tomaz Curk, Jeroen Krijgsveld, Matthias W. HentzeAbstract:This protocol is an extension to: Nat. Protoc. 8, 491-500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native Interactions with RNA. In brief, cells are irradiated with UV light to induce protein-RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein-RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supeRNAtant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown.
Aino I. Järvelin - One of the best experts on this subject based on the ideXlab platform.
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Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap
Nature Protocols, 2017Co-Authors: Alfredo Castello, Aino I. Järvelin, Bernd Fischer, Christian K. Frese, Rastislav Horos, Anne-marie Alleaume, Sophia Foehr, Tomaz Curk, Jeroen Krijgsveld, Matthias W. HentzeAbstract:This protocol is an extension to: Nat. Protoc. 8 , 491–500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013 RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native Interactions with RNA. In brief, cells are irradiated with UV light to induce protein–RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein–RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supeRNAtant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown. Here the authors provide an extension to their earlier RNA interactome capture protocol. This Protocol Extension describes RBDmap—a method to identify the regions of RNA-binding proteins engaged in native Interactions with RNA, in a proteome-wide manner.
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Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap
Nature protocols, 2017Co-Authors: Alfredo Castello, Aino I. Järvelin, Bernd Fischer, Christian K. Frese, Rastislav Horos, Anne-marie Alleaume, Sophia Foehr, Tomaz Curk, Jeroen Krijgsveld, Matthias W. HentzeAbstract:This protocol is an extension to: Nat. Protoc. 8, 491-500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native Interactions with RNA. In brief, cells are irradiated with UV light to induce protein-RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein-RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supeRNAtant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown.
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The new (dis)order in RNA regulation
Cell Communication and Signaling, 2016Co-Authors: Aino I. Järvelin, Marko Noerenberg, Ilan Davis, Alfredo CastelloAbstract:RNA-binding proteins play a key role in the regulation of all aspects of RNA metabolism, from the synthesis of RNA to its decay. Protein-RNA Interactions have been thought to be mostly mediated by canonical RNA-binding domains that form stable secondary and tertiary structures. However, a number of pioneering studies over the past decades, together with recent proteome-wide data, have challenged this view, revealing surprising roles for intrinsically disordered protein regions in RNA binding. Here, we discuss how disordered protein regions can mediate protein-RNA Interactions, conceptually grouping these regions into RS-rich, RG-rich, and other basic sequences, that can mediate both specific and non-specific Interactions with RNA. Disordered regions can also influence RNA metabolism through protein aggregation and hydrogel formation. Importantly, protein-RNA Interactions mediated by disordered regions can influence nearly all aspects of co- and post-transcriptional RNA processes and, consequently, their disruption can cause disease. Despite growing interest in disordered protein regions and their roles in RNA biology, their mechanisms of binding, regulation, and physiological consequences remain poorly understood. In the coming years, the study of these unorthodox Interactions will yield important insights into RNA regulation in cellular homeostasis and disease.
John T Lis - One of the best experts on this subject based on the ideXlab platform.
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comprehensive analysis of RNA protein Interactions by high throughput sequencing RNA affinity profiling
Nature Methods, 2014Co-Authors: Jacob M Tome, Abdullah Ozer, John M Pagano, Dan Gheba, Gary P Schroth, John T LisAbstract:The high-throughput sequencing–RNA affinity profiling (HiTS-RAP) assay enables large-scale profiling of protein Interactions with RNA libraries using a simple protocol on a high-throughput sequencer.
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a specific RNA hairpin loop structure binds the RNA recognition motifs of the drosophila sr protein b52
Molecular and Cellular Biology, 1997Co-Authors: Hua Shi, Bryan E Hoffman, John T LisAbstract:B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific Interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.
Masato Katahira - One of the best experts on this subject based on the ideXlab platform.
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Structure of the C-terminal RNA-binding domain of hnRNP D0 (AUF1), its Interactions with RNA and DNA, and change in backbone dynamics upon complex formation with DNA.
Journal of molecular biology, 2001Co-Authors: Masato Katahira, Takashi Nagata, Go Matsuda, Fuyuki Ishikawa, Youhei Miyanoiri, Yoshiaki Enokizono, Seiichi UesugiAbstract:Abstract Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP) -type RNA-binding domains (RBDs), each of which can specifically bind to the UUAG-sequence. hnRNP D0 also binds specifically to single-stranded d(TTAGGG) n , the human telomeric DNA repeat. We have already reported the structure and Interactions with RNA of the N-terminal RBD (RBD1). Here, the structure of the C-terminal RBD (RBD2) determined by NMR is presented. It folds into a compact αβ structure comprising an antiparallel β-sheet packed against two α-helices, which is characteristic of RNP-type RBDs. In addition to the four β-strands commonly found in RNP-type RBDs, an extra β-strand, termed β4 − , was found just before the fourth β-strand, yielding a five-stranded β-sheet. Candidate residues of RBD2 involved in the Interactions with RNA were identified by chemical shift perturbation analysis. Perturbation was detected on the β-sheet side, not on the opposite α-helix side, as observed for RBD1. It is notable that the β4 − to β4 region of RBD2 is involved in the Interactions in contrast to the case of RBD1. The chemical shift perturbation analysis also showed that RBD2 interacts with DNA in essentially the same way as with RNA. Changes in the backbone dynamics upon complex formation with DNA were examined by means of model free analysis of relaxation data. In free RBD2, the β4 − to β4 region exhibits slow conformational exchange on the milli- to microsecond time scale. The exchange is quenched upon complex formation. The flexibility of free RBD2 may be utilized in the recognition process by allowing different conformational states to be accessed and facilitating induced fit. Additionally, faster flexibility on the nano- to picosecond time scale was observed for loop 3 located between β2 and β3 in free RBD2, which is retained by the complex as well.
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Structure and Interactions with RNA of the N-terminal UUAG-specific RNA-binding domain of hnRNP D0.
Journal of molecular biology, 1999Co-Authors: Takashi Nagata, Yasuyuki Kurihara, Go Matsuda, Jun Ichi Saeki, Toshiyuki Kohno, Yasuko Yanagida, Fuyuki Ishikawa, Seiichi Uesugi, Masato KatahiraAbstract:Abstract Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), each of which can bind solely to the UUAG sequence specifically. The structure of the N-terminal RBD (RBD1) determined by NMR is presented here. It folds into a compact αβ structure comprising a four-stranded antiparallel β-sheet packed against two α-helices, which is characteristic of the RNP-type RBDs. Special structural features of RBD1 include N-capping boxes for both α-helices, a β-bulge in the second β-strand, and an additional short antiparallel β-sheet coupled with a β-turn-like structure in a loop. Two hydrogen bonds which restrict the positions of loops were identified. Backbone resonance assignments for RBD1 complexed with r(UUAGGG) revealed that the overall folding is maintained in the complex. The candidate residues involved in the Interactions with RNA were identified by chemical shift perturbation analysis. They are located in the central and peripheral regions of the RNA-binding surface composed of the four-stranded β-sheet, loops, and the C-terminal region. It is suggested that non-specific Interactions with RNA are performed by the residues in the central region of the RNA-binding surface, while specific Interactions are performed by those in the peripheral regions. It was also found that RBD1 has the ability to inhibit the formation of the quadruplex structure.
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STRUCTURE, BACKBONE DYNAMICS AND Interactions with RNA OF THE C-TERMINAL RNA-BINDING DOMAIN OF A MOUSE NEURAL RNA-BINDING PROTEIN, MUSASHI1
Journal of molecular biology, 1999Co-Authors: Takashi Nagata, Yasuyuki Kurihara, Seiichi Uesugi, Reisuke Kanno, Takao Imai, Shin Ichi Sakakibara, Hideyuki Okano, Masato KatahiraAbstract:Abstract Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in the regulation of asymmetric cell division. Musashi1 contains two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2. Our previous studies showed that RBD1 alone binds to RNA, while the binding of RBD2 is not detected under the same conditions. Joining of RBD2 to RBD1, however, increases the affinity to greater than that of RBD1 alone, indicating that RBD2 contributes to RNA-binding. We have determined the three-dimensional solution structure of the C-terminal RBD (RBD2) of Musashi1 by NMR. It folds into a compact αβ structure comprising a four-stranded antiparallel β-sheet packed against two α-helices, which is characteristic of RNP-type RBDs. Special structural features of RBD2 include a β-bulge in β2 and a shallow twist of the β-sheet. The smaller 1 H- 15 N nuclear Overhauser enhancement values for the residues of loop 3 between β2 and β3 suggest that this loop is flexible in the time-scale of nano- to picosecond order. The smaller 15 N T 2 values for the residues around the border between α2 and the following loop (loop 5) suggest this region undergoes conformational exchange in the milli- to microsecond time-scale. Chemical shift perturbation analysis indicated that RBD2 binds to an RNA oligomer obtained by in vitro selection under the conditions for NMR measurements, and thus the nature of the weak RNA-binding of RBD2 was successfully characterized by NMR, which is otherwise difficult to assess. Mainly the residues of the surface composed of the four-stranded β-sheet, loops and C-terminal region are involved in the interaction. The appearance of side-chain NH proton resonances of arginine residues of loop 3 and imino proton resonances of RNA bases upon complex formation suggests the formation of intermolecular hydrogen bonds. The structural arrangement of the rings of the conserved aromatic residues of β2 and β3 is suitable for stacking interaction with RNA bases, known to be one of the major protein-RNA Interactions, but a survey of the perturbation data suggested that the stacking interaction is not ideally achieved in the complex, which may be related to the weaker RNA-binding of RBD2.