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Genetically encoded chemical crosslinking of RNA in vivo

Abstract

Protein–RNA interactions regulate RNA fate and function, and defects can lead to various disorders. Such interactions have mainly been studied by nucleoside-based UV crosslinking methods, which lack broad in vivo compatibility and the ability to resolve specific amino acids. In this study we genetically encoded latent bioreactive unnatural amino acids into proteins to react with bound RNA by proximity-enabled reactivity and demonstrated genetically encoded chemical crosslinking of proteins with target RNA (GECX-RNA) in vivo. Applying GECX-RNA to the RNA chaperone Hfq in Escherichia coli identified target RNAs with amino acid specificity. Combining GECX-RNA with immunoprecipitation and high-throughput sequencing of an N6-methyladenosine reader protein in mammalian cells allowed the in vivo identification of unknown N6-methyladenosine on RNA with single-nucleotide resolution throughout the transcriptome. GECX-RNA thus affords resolution at the nucleotide and amino acid level for interrogating protein–RNA interactions in vivo. It also enables the precise engineering of covalent linkages between a protein and RNA, which will inspire innovative solutions for RNA-related research and therapeutics.

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Fig. 1: GECX-RNA enables FSY-incorporated dPsCas13b to crosslink target RNA in vitro.
Fig. 2: GECX-RNA enables FSY-incorporated Hfq proteins to crosslink target RNA in E. coli.
Fig. 3: GECX-RNA enables FSY-incorporated dPsCas13b proteins to crosslink target RNA in mammalian cells.
Fig. 4: Genetically encoding SFY allows crosslinking of His, Tyr and Lys residues in protein and of RNA in cells.
Fig. 5: Design of GRIP-seq for in vivo detection of m6A on RNA with single-nucleotide resolution.
Fig. 6: GRIP-seq in vivo detected m6A on RNA with single-nucleotide resolution in mammalian cells.

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Data availability

All GRIP-seq data are available in the Sequence Read Archive through accession number PRJNA797913. All other data generated or analysed in this study are available within the article and its Supplementary Information. Source data are provided with this paper.

Code availability

The custom code used in this study is available at https://github.com/Shall-We-Dance/GRIP-seq.

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Acknowledgements

L.W acknowledges the support of the NIH (R01GM118384 and R01CA258300). Y.S. acknowledges the support of the NIH (R01AG057497 and R01EY027789).

Author information

Authors and Affiliations

Authors

Contributions

W.S. designed and conducted the experiments, analysed the data and wrote the manuscript; N.W. evolved SFYRS and characterized SFY incorporation and the crosslinking of proteins; H.L. conducted the data analysis of GRIP-seq; B.Y. synthesized FSY and SFY, performed the SFY reactions with NMPs in vitro and analysed the data; L.J. helped with the dCas13b target RNA crosslinking and enrichment in mammalian cells; X.R. and Y.S. helped with the GRIP-seq experiments; L.W. conceived, directed and supported the project and wrote the manuscript.

Corresponding author

Correspondence to Lei Wang.

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The authors declare no competing interests.

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Nature Chemistry thanks Ryan Flynn, Stephen Fried and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Identification of the positively charged residues of PsCas13b involved in pre-crRNA cleavage.

a) Multiple sequence alignment of Cas13b proteins from different species (Bzo: Bergeyella zoohelcum, Psp: Prevotella sp. P5-125, Pgu: Porphyromonas gingivalis, Pbu: Prevotella buccae, and Ran: Riemerella anatipestifer) for β-sheets 5 and 6 involved in pre-crRNA cleavage. The secondary structure of BzoCas13b is shown above the sequence28. Identical and similar residues are highlighted in red and white boxes, respectively. Positive charged catalytic residues in BzoCas13b involved in the pre-crRNA cleavage on β-sheets 5 and 6 (450 R, 452 K, 459 R) are marked with green stars on the bottom. Positive charged residues in PsCas13b located on β-sheets 5 and 6 (367 K, 370 K, 378 R, 380 R) are marked with purple squares. Multiple sequence alignment of full-length Cas13b proteins from different species is shown in Supplementary Fig. 1. b) Denaturing urea-PAGE demonstrating the pre-crRNA cleavage by dPsCas13b-WT and dPsCas13b-Ala-mutants speculatively involved in the pre-crRNA processing. dPsCas13b-WT and dPsCas13b-Ala-mutants were incubated with pre-crRNA and then separated on denaturing urea-PAGE. The Urea-gel was stained with SybrGold for fluorescent detection of RNA.

Source data

Extended Data Fig. 2 GRIP results demonstrate that site 25 of Hfq directly binds with (AAN)4 elements of rpoS RNA.

GRIP identified the binding sites of Tyr25 of Hfq on rpoS RNA in E. coli cells. Red triangles indicate cross-linking sites identified from GRIP for rpoS RNA from Hfq-25FSY expressing E. coli cells. Two examples of Sanger sequencing of clones from Hfq-25FSY sample were shown below.

Source data

Extended Data Fig. 3 Examples of GRIP-seq data for m6A identification.

Genome browser tracks of GRIP-seq data in JUN and DICER1 mRNA regions. Reverse-transcription-termination sites (RT-termination sites) from GRIP-seq were marked as yellow triangles. Known m6A sites from published datasets were marked as grey triangles.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7, Tables 1–4 and source data files for the supplementary figures.

Reporting Summary

Supplementary Table 2

Sequencing statistics for GRIP-seq.

Supplementary Table 3

m6A sites identified from GRIP-seq (coordinates in hg19 human genome assembly).

Supplementary Data 1

Statistical source data for Supplementary Fig. 2b.

Supplementary Data 2

Statistical source data for Supplementary Fig. 4.

Supplementary Data 3

Statistical source data for Supplementary Fig. 5.

Supplementary Data 4

Source data for Sanger sequencing in Supplementary Fig. 2d (Ab1 files).

Supplementary Data 5

Source data for Sanger sequencing in Supplementary Fig. 2g (Ab1 files).

Supplementary Data 6

Source data for Sanger sequencing in Supplementary Fig. 7h (Ab1 files).

Source data

Source Data Fig. 1

Unprocessed gels.

Source Data Fig. 2

Unprocessed western blot and statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Uncropped images, unprocessed gels, unprocessed western blots and statistical source data.

Source Data Extended Data Fig. 1

Unprocessed gels.

Source Data Extended Data Fig. 2

Source data for Sanger sequencing (Ab1 files).

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Sun, W., Wang, N., Liu, H. et al. Genetically encoded chemical crosslinking of RNA in vivo. Nat. Chem. 15, 21–32 (2023). https://doi.org/10.1038/s41557-022-01038-4

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