Structural modeling of protein–RNA complexes using crosslinking of segmentally isotope-labeled RNA and MS/MS

Dorn, Georg; Leitner, Alexander; Boudet, Julien; Campagne, Sébastien; Schroetter, Christine von; Moursy, Ahmed; Aebersold, Ruedi; Allain, Frédéric H.‐T.

doi:10.1038/nmeth.4235

Cited by 52 publications

(142 citation statements)

References 45 publications

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“…Given the importance of the added C-terminal helix in the RBM20 RRM domain, we searched other RRM domains that may use the same RNA-binding mechanism. The only candidate we found was the first RRM domain of polypyrimidine tract-binding protein (PTBP1/PTB4) bound to a viral internal ribosome entry site (vIRES) stemloop RNA (PDB ID 2N3O; (Dorn et al, 2017). A sequence similarity was also previously identified in the RNP1 motifs of RBM20 and RRM1 of PTBP1 (Filippello et al, 2013).…”

Section: Structural Similarity To Polypyrimidine Tract-binding Proteinmentioning

confidence: 85%

“…However, the overall affinity for both is relatively moderate (KD values of 5.7 and 8 μM, respectively). It is possible that RRM1 from PTBP1 exhibits a preference for UCUU in a particular context (Dorn et al, 2017), in which case PTBP1 binding would relegate RBM20 binding to the remaining UCUU sites.…”

Section: In Terms Of Protein-protein Interactions Polypyrimidine Tramentioning

confidence: 99%

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Structural basis of UCUU RNA motif recognition by splicing factor RBM20

Upadhyay

Mackereth

2019

Preprint

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The vertebrate splicing factor RBM20 (RNA Binding Motif protein 20) regulates protein isoforms important for heart development and function, with mutations in the gene linked to cardiomyopathy. Previous studies have identified the four base RNA motif UCUU as a common element in pre-mRNA targeted by RBM20. Here, we have determined the structure of the RNA Recognition Motif (RRM) domain from mouse RBM20 bound to RNA containing a UCUU sequence. The atomic details show that the RRM domain spans a larger region than initially proposed in order to interact with the complete UCUU motif, with a well-folded Cterminal helix encoded by exon 8 critical for high affinity binding. This helix only forms upon binding RNA with the final uracil, and removing the helix reduces affinity as well as specificity. We therefore find that RBM20 uses a coupled folding-binding mechanism by the C-terminal helix to specifically recognize the UCUU RNA motif.

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Section: Structural Similarity To Polypyrimidine Tract-binding Proteinmentioning

confidence: 85%

Section: In Terms Of Protein-protein Interactions Polypyrimidine Tramentioning

confidence: 99%

Structural basis of UCUU RNA motif recognition by splicing factor RBM20

Upadhyay

Mackereth

2019

Preprint

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“…10,12,13 Besides, we anticipate a potential use of the large SI labelled RNAs in recently reported mass spectrometric methods to localize protein binding sites, which give valuable information for the 3D structure modelling of large RNP particles. 31,32 We currently also focus on the synthesis of per-deuterated RNA building blocks. The building blocks will be beneficial for NMR but also for SAXS/SANS studies in an integrative structural biology approach, as the chemical RNA synthesis allows full control over segmental deuteration.…”

Section: -13mentioning

confidence: 99%

Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA

et al. 2017

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We showcase the high potential of the 2 0 -cyanoethoxymethyl (CEM) methodology to synthesize RNAs with naturally occurring modified residues carrying stable isotope (SI) labels for NMR spectro- Solution and solid state nuclear magnetic resonance (NMR) spectroscopy have proven to be highly suitable to address structural and dynamic features of RNA. 1-4 A prerequisite to apply state-of-the-art NMR experiments is the introduction of a stable isotope (SI) labelling pattern using 13 C/ 15 N labelled RNA or DNA precursors. [5][6][7][8] The most wide-spread method uses labelled (2 0 -deoxy)-ribonucleotide triphosphates and enzymes to produce the desired RNA or DNA sequence enriched with 13 C and 15 N nuclei. 1,5 This approach enables to produce sufficient amounts of RNA and DNA for NMR spectroscopic applications. This well-established method allows nucleotide specific labeling by mixing a SI-labeled with unlabeled d/rNTPs. Especially in larger RNAs (460 nt) such nucleotide specific SI-labeling can still lead to significant resonance overlap. That is why, the PLOR (position-selective labelling of RNA) method was recently introduced, which holds the promise to site-specifically label RNA using SI-labelled ribonucleotide triphosphates and T7 RNA polymerase. 9 An alternative method was concurrently developed making use of the synthesis of 2 0 -O-tri-iso-propylsilyloxymethylphosphoramidites and solid phase synthesis. 10-13The approach works well for medium sized RNAs up to 50 nts and the synthetic access to the SI-labelled building blocks is well established.10,12 Thus, the fully chemical SI-labelling protocol can be regarded as an expedient expansion to the settled enzymatic procedures to freely chose the number and positioning of SI-labeled residues into a target RNA. In our hands, however, the standard solid phase synthesis methods are not that well suited to produce larger amounts (450 nmol) and purities higher than 95% for RNAs exceeding 60 nts. Due to this restriction, large RNAs are only accessible via enzymatic ligation strategies using T4 RNA/DNA ligase making extra optimization steps necessary or introducing new problems, such as finding the optimal ligation site or issues regarding up-scaling and yield of the ligation product. 14-16 Thus, an improved synthetic procedure to directly address SI-labelling of larger RNAs (460 nt) at amounts suitable for NMR would be highly desirable. We report the synthesis of SI-labelled RNAs ranging in size between 60 to 80 nts capitalizing on the 2 0 -cyanoethoxymethyl (CEM) RNA synthesis method. 17,18 As these CEM building blocks are not commercially available all phosphoramidites were produced in-house and we further synthesized 13 C-/ 15 N-labelled unmodified and naturally occurring modified RNA phosphoramidites (Fig. 1a and b). In detail, we focused on the synthesis of 8- We used these monomer units to produce SI-labelled RNAs exceeding the size limitation of 60 nucleotides for NMR up to 20 nucleotides. The RNAs reported here were synthesized on a 1.3 mmol scale and on a 1000...

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“…Concurrently, chemical cross‐linking/mass spectrometry (XLMS) is an experimental strategy that is gaining momentum in the structural biology community . It relies on the exposure of protein structures to reactants (the cross‐linkers) which react to pairs of residues, forming cross‐links.…”

Section: Introductionmentioning

confidence: 99%

“…Concurrently, chemical cross-linking/mass spectrometry (XLMS) is an experimental strategy that is gaining momentum in the structural biology community. [11][12][13][14] It relies on the exposure of protein structures to reactants (the cross-linkers) which react to pairs of residues, forming cross-links. The identification of the amino acid pairs crosslinked is performed with high-resolution mass spectrometry, providing a distance constraint that can be used to aid protein structure modeling.…”

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confidence: 99%

Structural complementarity of distance constraints obtained from chemical cross‐linking and amino acid coevolution

et al. 2019

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The analysis of amino acid coevolution has emerged as a practical method for protein structural modeling by providing structural contact information from alignments of amino acid sequences. In parallel, chemical cross‐linking/mass spectrometry (XLMS) has gained attention as a universally applicable method for obtaining low‐resolution distance constraints to model the quaternary arrangements of proteins, and more recently even protein tertiary structures. Here, we show that the structural information obtained by XLMS and coevolutionary analysis are effectively complementary: the distance constraints obtained by each method are almost exclusively associated with non‐coincident pairs of residues, and modeling results obtained by the combination of both sets are improved relative to considering the same total number of constraints of a single type. The structural rationale behind the complementarity of the distance constraints is discussed and illustrated for a representative set of proteins with different sizes and folds.

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Structural modeling of protein–RNA complexes using crosslinking of segmentally isotope-labeled RNA and MS/MS

Cited by 52 publications

References 45 publications

Structural basis of UCUU RNA motif recognition by splicing factor RBM20

Structural basis of UCUU RNA motif recognition by splicing factor RBM20

Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA

Structural complementarity of distance constraints obtained from chemical cross‐linking and amino acid coevolution

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