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RNA molecular recording with an engineered RNA deaminase

Nature Methods volume 20, pages 1887–1899 (2023)Cite this article

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Abstract

RNA deaminases are powerful tools for base editing and RNA molecular recording. However, the enzymes used in currently available RNA molecular recorders such as TRIBE, DART or STAMP have limitations due to RNA structure and sequence dependence. We designed a platform for directed evolution of RNA molecular recorders. We engineered an RNA A-to-I deaminase (an RNA adenosine base editor, rABE) that has high activity, low bias and low background. Using rABE, we present REMORA (RNA-encoded molecular recording in adenosines), wherein deamination by rABE writes a molecular record of RNA–protein interactions. By combining rABE with the C-to-U deaminase APOBEC1 and long-read RNA sequencing, we measured binding by two RNA-binding proteins on single messenger RNAs. Orthogonal RNA molecular recording of mammalian Pumilio proteins PUM1 and PUM2 shows that PUM1 competes with PUM2 for a subset of sites in cells. Furthermore, we identify transcript isoform-specific RNA–protein interactions driven by isoform changes distal to the binding site. The genetically encodable RNA deaminase rABE enables single-molecule identification of RNA–protein interactions with cell type specificity.

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Fig. 1: Screening for an ssRNA adenosine deaminase.
Fig. 2: Engineering an improved ssRNA adenosine deaminase by directed protein evolution.
Fig. 3: Transcriptome-wide characterization of rABE.
Fig. 4: rABE recovers known binding patterns of Pumilio proteins.
Fig. 5: PacBio long-read sequencing identifies isoform-specific binding events of PUM1.
Fig. 6: Co-occupancy of PUM1 and PUM2 on RNA using rABE and APOBEC1 fusions.

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Data availability

Plasmids encoding rABE are available through Addgene (191383–191386). Sequencing data are available at GEO accession number GSE216334. Source data are provided with this paper.

Code availability

Analysis code is available at https://github.com/linyz/remora.

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Acknowledgements

The authors thank members of the Floor laboratory for feedback on this work and their continued support. Computation was supported by the UCSF Wynton high-performance computing infrastructure, PacBio sequencing was supported through instrumentation shared with V. Ramani (Gladstone Institutes), high-throughput short-read sequencing was supported by the UCSF Center for Advanced Technology, and flow cytometry was supported by the Parnassus Flow Cytometry Core at UCSF. This work was supported by the UCSF Program for Breakthrough Biomedical Research, funded in part by the Sandler Foundation (to S.N.F.) and the National Institutes of Health DP2GM132932 and R35GM149255 (to S.N.F.). S.N.F. is a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts.

Author information

Author notes

  1. Bao Quoc Thai

    Present address: MSTP Program, University of Arizona, Tuscon, AZ, USA

Authors and Affiliations

  1. Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA

    Yizhu Lin, Samentha Kwok, Bao Quoc Thai & Stephen N. Floor

  2. Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA, USA

    Abigail E. Hein

  3. Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA

    Yewande Alabi

  4. Gladstone Institute for Data Science and Biotechnology, San Francisco, CA, USA

    Megan S. Ostrowski & Ke Wu

  5. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA

    Stephen N. Floor

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Contributions

Conceptualization, Y.L. and S.N.F.; Investigation, Y.L., S.K., B.Q.T., S.N.F. and Y.A.; Writing – Original Draft, Y.L. and S.N.F.; Writing – Review & Editing, Y.L. and S.N.F.; Methodology, Y.L. and S.N.F.; Resources, M.S.O. and K.W.; Formal analysis, Y.L.; Software, Y.L. and A.E.H.; Funding Acquisition, S.N.F.; Supervision, S.N.F.

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Correspondence to Stephen N. Floor.

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Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editors: Rita Strack and Lei Tang, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 Design of eGFP-UAG-BoxB-mCherry fluorescent reporter.

RNA secondary structure modeling around UAG-BoxB cassette with RNAfold. BoxB forms a stable stem-loop structure, while the UAG stop codon was predicted to be unstructured.

Extended Data Fig. 2 Directed evolution for an improved ssRNA A-to-I editor.

(a) OD600 measurement of E. coli growth curve after transformation of ABE7.10 under different kanamycin concentrations. 0.375 x concentration (18.75 mg/L) was the optimal concentration that allowed growth of ABE.10 expressing cells and inhibited growth of ABE7.10-E59A negative control cells. (b) Scatter plots of flow cytometry measurement of the editing efficiency of ABE7.10 and ABE7.10-C146S. (c) Western blot of ABE variants in Fig. 2d, ABE7.10 had low expression and was not detected. Expression level and fluorescent reporter assay for rABE were independently tested at least three times, with consistent results. (d) Structural model of the S. aureus tadA protein near Thr145 (corresponding to E. coli Ser146). (e) Sanger sequencing measurement of A-to-I editing rates introduced by ABE7.10 and ABE7.10-C146S on an adenosine-rich UAG-BoxB reporter. Several A-to-I editing were observed flanking both sides of BoxB in different sequence contexts.

Source data

Extended Data Fig. 3 Transcriptome-wide characterization of rABE.

(a) Cell proliferation of wt HEK293T cells and doxycycline-inducible rABE-Flag expressing HEK293T cells measured by cell counts. Cells were incubated with 0, 50, or 1000 ng/mL doxycycline for 3 days. Error bars are 95% CI around mean. n = 3 biologically independent samples. (b) Cell proliferation measured by CFSE labeling of HEK293T stable cell lines expressing rABE under 0, 50, 1000 ng/mL doxycycline. Error bars are 95% CI around mean. n = 3 biologically independent samples. (c, d) Scatter plots of significant A-to-I editing events induced by Rbfox2-rABE under 50 ng/mL (C) and 1000 ng/mL (D) after 12, 24 and 48 hours. (e) Scatter plot of Rbfox2-rABE-induced A-to-I editing rate, comparing 24 hours incubation vs 12 hours incubation of 1000 ng/mL doxycycline; 48 hours incubation vs 24 incubation of 1000 ng/mL doxycycline; and 1000 ng/mL vs 50 ng/mL doxycycline after incubation for 48 hours. (f) Western blot of Rbfox2-hyperTRIBE and endogenous Rbfox2 in HEK293T doxycycline-inducible cell lines, 4 biologically independent replicates each. (g) Western blot of Rbfox2-rABE and endogenous Rbfox2 in HEK293T doxycycline-inducible cell lines, 4 biologically independent replicates each. (h) Western blot measurement of rABE, APOBEC1, Rbfox2-rABE, Rbfox2-APOBEC1 expression levels under 1000 ng/mL doxycycline condition, 3 replicates each. All proteins contain a Flag tag at their C terminus and are blotted with anti-Flag-HRP. (i, j) Differential expression analysis comparing transcriptome-wide gene expression level after doxycycline induction (1000 ng/mL vs no doxycycline control) of rABE, hyperTRIBE and APOBEC1 (I) and of Rbfox2-rABE, Rbfox2-hyperTRIBE and Rbfox2-APOBEC1 (J). Expression of rABE or APOBEC1 alone and their Rbfox2 fusions do not change gene expression levels, while hyperTRIBE induced more genes with expression level changes. (k) CentriMo analysis around Rbfox2-APOBEC1 induced C-to-U editing sites. (l) In vitro structure probing icSHAPE scores around editing sites. Rbfox2-hyperTRIBE and hyperTRIBE showed lower SHAPE reactivity around the editing sites, and lower SHAPE reactivity is a signal of increased structure. (m) Distribution of rABE, hyperTRIBE and APOBEC1 background editing sites across transcript regions. (n) Nucleotide composition at −1 or +1 position of editing sites. Y-axes are log2 fold change or expected value (0.25). (o) Distribution of Rbfox2-rABE, Rbfox2-hyperTRIBE and Rbfox2-APOBEC1 editing sites across transcript regions.

Source data

Extended Data Fig. 4 rABE recovers known binding patterns of Pumilio proteins.

(a) Venn diagram comparison of PUM1-rABE targets (this study) and PUM1 targets identified by CLIP-seq (Sternburg et al.37). (b) Venn diagram comparison of PUM1-rABE targets and PUM2-rABE targets. (c) Venn diagram comparison of PUM2-rABE targets (this study) and PUM2 targets identified by CLIP-seq (Sternburg et al.37). (d) De novo motif discovery in PUM1-rABE hits that are also identified by CLIP-seq or not identified by CLIP-seq. Both populations successfully recovered the known PBE motif UGUANA. (e) PUM1 target genes identified by CLIP-seq but not PUM1-rABE showed significantly lower expression level (p < 2.2e-16, t-test, two-sided, n = 3322 for CLIP only group, n = 879 for CLIP and PUM1-rABE group). Centre: median; box bounds: 25% and 75% percentile; whisker: 1.5x of interquartile range. All outliers are marked as dots. (f) Western blot of PUM1-rABE and endogenous PUM1 in HEK293T dox inducible cell lines, 4 independent biological replicates each. (g) Western blot of PUM2-rABE and endogenous PUM2 in HEK293T dox inducible cell lines, 4 independent biological replicates each. (h) Scatter plot showing editing rate in PUM1-rABE cell lines, under 50 or 1000 ng/mL doxycycline conditions.

Source data

Extended Data Fig. 5

PUM1-APOBEC1 and PUM2-APOBEC1 induced editing patterns in the 3′ UTR region of UBB and PCNA 3′ UTR regions.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Supplementary Tables 1–4.

Reporting Summary

Peer Review File

Supplementary Table 5

Summary of isoform-specific PUM1 binding events.

Source data

Source Data Fig. 1

Statistical source data table for Fig. 1 c,d,e,g.

Source Data Fig. 2

Statistical source Data table for Fig. 2c,d.

Source Data Fig. 3

Statistical source data table for Fig. 3f,g,h,m,n.

Source Data Fig. 4

Statistical source data table for Fig. 4g,h.

Source Data Fig. 5

Statistical source data table for Fig. 5b,c,f,k,l,m.

Source Data Fig. 6

Statistical source data table for Fig. 6d,e,g,h.

Source Data Extended Data Fig./Table 2

Source data table for Extended Data Fig. 2a.

Source Data Extended Data Fig./Table 3

Source data table for Extended Data Fig. 3a,b,i.

Source Data Extended Data Fig./Table 4

Source statistical data table for Extended Data Fig. 4e.

41592_2023_2046_MOESM14_ESM.pdf

Source Data Extended Data Fig./Table 2. Unprocessed western blots for Extended Data Fig. 2c. Source Data Extended Data Fig./Table 3. Unprocessed western blots for Extended Data Fig. 3f,g. Source Data Extended Data Fig./Table 4. Unprocessed western blots for Extended Data Fig. 4f,g.

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Lin, Y., Kwok, S., Hein, A.E. et al. RNA molecular recording with an engineered RNA deaminase. Nat Methods 20, 1887–1899 (2023). https://doi.org/10.1038/s41592-023-02046-z

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