Photocrosslinking between nucleic acids and proteins: general discussion

Bhat, Vinayak; Cogdell, Richard J.; Crespo‐Hernández, Carlos E.; Datta, Ankona; De, Arijit; Haacke, S.; Helliwell, John R.; Improta, Roberto; Joseph, Joshy; Karsili, Tolga N. V.; Kohler, Bern; Krishnan, Retheesh; Mahil, L; Lewis, Frederick D.; Mandal, Imon; Markovitsi, Dimitra; Mishra, Padmaja P.; Paul, Sneha; Periyasamy, Ganga; Pradeepkumar, P. I.; Chowdhury, Priyadarshi Roy; Sarangi, Manas Kumar; Sasikumar, Devika; Schapiro, Igor; Schertler, Gebhard F. X.; Schlichting, Ilme; Segarra‐Martí, Javier; Swaminathan, Rajaram; V, Vishnu; Grondelle, Rienk van; Varghese, Reji; Venkatramani, Ravindra

doi:10.1039/c8fd90005a

“…Other researchers have noted in passing the increased presence of aromatic amino acid side chains in UV cross-linking data sets (see refs 18,19,33,48,61 ), but have not to our knowledge recognized its role as a trigger for cross-linking, nor distinguished between direct and indirect cross-link events. It is clear that additional factors may also contribute to cross-linking events in RNA-protein sites, including: efficiency of the photo-induced electron transfer, the ability to stabilize free radicals, the flexibility of the system to adopt to the configurations that are required for the radical reactions 62 and the proximity of reactive pairs 63 (Sarnowski et al; in preparation). Furthermore, our findings cannot explain all RNA-protein cross-linking reactions, at least those involving sulphurcontaining amino acids, such as cysteine, which are highly photoreactive and prone to cross-link probably due to the high reactivity of the thiyl radical 19,34,55 .…”

Section: Discussionmentioning

confidence: 99%

Structural requirements for photo-induced RNA-protein cross-linking

Knörlein

¹

,

Sarnowski

²

,

Vries

³

et al. 2021

Preprint

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Understanding structure-function relationships of RNA-binding proteins requires knowledge of how they bind RNAs in vivo. RNA-protein interactions are studied using light-induced cross-linking at "zerodistance", yielding nucleotide/amino-acid adducts for mass-spectrometry (MS)-based characterization.However, prerequisites for cross-linking are poorly understood, limiting interpretation of cross-linking data. Here, we report novel insights on cross-linking requirements from studying RBFOX-RRM domain bound to 13 C-labeled variants of its heptaribonucleotide binding element as a model. We probed the influence of nucleotide identity, sequence position and amino-acid composition using tandem-MS to assign cross-links at site-specific resolution. We observed cross-linking at three nucleotides, which were stacked onto phenylalanines. Surprisingly, this stacking was required for neighbouring aminoacids to cross-link, and is apparent in published RNA-protein datasets. We hypothesize that πstacking activates cross-linking via electron transfer, whereafter nucleotide-and peptide radicals, possibly stabilized by capto-dative effects, recombine. These findings should facilitate interpretation of cross-linking data from structural studies and genome-wide datasets. has also been observed with proteins that lack canonical RNA binding domains (RBDs) 1 . Taken together, these features render difficult the prediction of an RBP's substrates based only on a computational search for its consensus RBE. Indeed, recent studies of the RBFOX protein family showed that only one half of the isolated RNA targets contain the RBFOX consensus binding motif and that other motifs presumably bear responsibility for some of its splicing activities [10][11][12] .Many state-of-the-art methods to identify RNA-protein interactions in vivo employ RNA-protein crosslinking induced by UV light [13][14][15][16] . For example, by combining UV cross-linking with mass spectrometry approaches, proteins bound to given RNAs can be identified [17][18][19][20][21][22][23][24] . Conversely, UV cross-linking and immunoprecipitation (CLIP) and related protocols are commonly used to identify RNA-binding sites for given proteins on a transcriptome-wide scale [25][26][27][28][29][30] . Technical advances constantly improve these techniques 18,31,32 , however, a long-standing challenge in structure/mechanism-oriented studies is to identify the points of cross-linking on both the RNA and the protein with site-specific resolution.Recently, we introduced cross-linking of segmentally isotope-labelled RNA and tandem mass spectrometry (CLIR-MS), which identifies the sites of amino acid/ribonucleotide cross-links in a single protocol 32 . In CLIR-MS, RNA regions that are suspected to interact with a protein are synthesized in isotopically labelled light-and heavy variants, so that nucleotides involved in cross-linking events appear as peak doublets in the resulting mass spectrum. By focusing on peak doublets during data analysis, cross-linked amino acids and nucleotides are reliabl...

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“…Other researchers have noted in passing the increased presence of aromatic amino acid side chains in UV cross-linking data sets (see refs 18,19,33,48,61 ), but have not to our knowledge recognized its role as a trigger for cross-linking, nor distinguished between direct and indirect cross-link events. It is clear that additional factors may also contribute to cross-linking events in RNA-protein sites, including: efficiency of the photo-induced electron transfer, the ability to stabilize free radicals, the flexibility of the system to adopt to the configurations that are required for the radical reactions 62 and the proximity of reactive pairs 63 (Sarnowski et al; in preparation). Furthermore, our findings cannot explain all RNA-protein cross-linking reactions, at least those involving sulphurcontaining amino acids, such as cysteine, which are highly photoreactive and prone to cross-link probably due to the high reactivity of the thiyl radical 19,34,55 .…”

Section: Discussionmentioning

confidence: 99%

Structural requirements for photo-induced RNA-protein cross-linking

Knörlein

¹

,

Sarnowski

²

,

Vries

³

et al. 2021

Preprint

View full text Add to dashboard Cite

Understanding structure-function relationships of RNA-binding proteins requires knowledge of how they bind RNAs in vivo. RNA-protein interactions are studied using light-induced cross-linking at “zero-distance”, yielding nucleotide/amino-acid adducts for mass-spectrometry (MS)-based characterization. However, prerequisites for cross-linking are poorly understood, limiting interpretation of cross-linking data. Here, we report novel insights on cross-linking requirements from studying RBFOX-RRM domain bound to 13C-labeled variants of its heptaribonucleotide binding element as a model. We probed the influence of nucleotide identity, sequence position and amino-acid composition using tandem-MS to assign cross-links at site-specific resolution. We observed cross-linking at three nucleotides, which were stacked onto phenylalanines. Surprisingly, this stacking was required for neighbouring amino-acids to cross-link, and is apparent in published RNA-protein datasets. We hypothesize that π-stacking activates cross-linking via electron transfer, whereafter nucleotide- and peptide radicals, possibly stabilized by capto-dative effects, recombine. These findings should facilitate interpretation of cross-linking data from structural studies and genome-wide datasets.

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“…In line with previous studies, it was found that G -quadruplexes exhibit a larger propensity than duplexes to photoeject an electron upon absorption of low energy photons. One hypothesis suggested previously is that electron ejection occurs after population of excited charge transfer states involving different bases, followed by charge separation [74]. According to such a scenario, the guanine core should behave as a deep trap for the positive charge, the negative charge remaining on an external base (adenine or thymine).…”

Section: Discussionmentioning

confidence: 99%

Populations and Dynamics of Guanine Radicals in DNA strands—Direct versus Indirect Generation

Balanikas

¹

,

Bányász

²

,

Baldacchino

³

et al. 2019

Self Cite

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Guanine radicals, known to be involved in the damage of the genetic code and aging, are studied by nanosecond transient absorption spectroscopy. They are generated in single, double and four-stranded structures (G-quadruplexes) by one and two-photon ionization at 266 nm, corresponding to a photon energy lower than the ionization potential of nucleobases. The quantum yield of the one-photon process determined for telomeric G-quadruplexes (TEL25/Na+) is (5.2 ± 0.3) × 10−3, significantly higher than that found for duplexes containing in their structure GGG and GG sequences, (2.1 ± 0.4) × 10−3. The radical population is quantified in respect of the ejected electrons. Deprotonation of radical cations gives rise to (G-H1)• and (G-H2)• radicals for duplexes and G-quadruplexes, respectively. The lifetimes of deprotonated radicals determined for a given secondary structure strongly depend on the base sequence. The multiscale non-exponential dynamics of these radicals are discussed in terms of inhomogeneity of the reaction space and continuous conformational motions. The deviation from classical kinetic models developed for homogeneous reaction conditions could also be one reason for discrepancies between the results obtained by photoionization and indirect oxidation, involving a bi-molecular reaction between an oxidant and the nucleic acid.

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Photocrosslinking between nucleic acids and proteins: general discussion

Cited by 7 publications

References 1 publication

Structural requirements for photo-induced RNA-protein cross-linking

Structural requirements for photo-induced RNA-protein cross-linking

Structural requirements for photo-induced RNA-protein cross-linking

Populations and Dynamics of Guanine Radicals in DNA strands—Direct versus Indirect Generation

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