Cell-type specific sequencing of microRNAs from complex animal tissues

Abstract

MicroRNAs (miRNAs) play an essential role in the post-transcriptional regulation of animal development and physiology. However, in vivo studies aimed at linking miRNA function to the biology of distinct cell types within complex tissues remain challenging, partly because in vivo miRNA-profiling methods lack cellular resolution. We report microRNome by methylation-dependent sequencing (mime-seq), an in vivo enzymatic small-RNA-tagging approach that enables high-throughput sequencing of tissue- and cell-type-specific miRNAs in animals. The method combines cell-type-specific 3′-terminal 2′-O-methylation of animal miRNAs by a genetically encoded, plant-specific methyltransferase (HEN1), with chemoselective small-RNA cloning and high-throughput sequencing. We show that mime-seq uncovers the miRNomes of specific cells within Caenorhabditis elegans and Drosophila at unprecedented specificity and sensitivity, enabling miRNA profiling with single-cell resolution in whole animals. Mime-seq overcomes current challenges in cell-type-specific small-RNA profiling and provides novel entry points for understanding the function of miRNAs in spatially restricted physiological settings.

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Figure 1: The methyltransferase HEN1 from Arabidopsis thaliana methylates animal miRNAs in vivo.
Figure 2: Periodate-mediated oxidation enables selective cloning of animal miRNAs upon restrictive Ath-HEN1-directed methylation.
Figure 3: Mime-seq reproducibly recovers neuronal miRNAs in C. elegans.
Figure 4: Mime-seq unveils the miRNomes of diverse tissues in C. elegans.
Figure 5: Mime-seq has the sensitivity to retrieve miRNAs from 1/300 cells.

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Acknowledgements

We thank Cochella lab members for various fosmid reporters; F. Schnorrer (IBDM, Marseille, France) for muscle-specific Gal4 driver lines; and the CGC (NIH-P40-OD010440) for worm strains. This work was supported by FP7/2007-2013 grants from the European Research Council to L.C. (ERC-StG-337161) and S.L.A. (ERC-StG-338252) and the Austrian Science Fund to L.C. (W-1207-B09 and SFB-F43-23) and S.L.A. (Y-733-B22 START, W-1207-B09 and SFB-F43-22). R.A.M. is a recipient of a DOC fellowship of the Austrian Academy of Sciences at IMBA. Basic research at IMP is supported by Boehringer Ingelheim GmbH.

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Contributions

S.L.A. conceived the strategy. L.C. and S.L.A. designed and supervised the study. C.A. performed and analyzed all C. elegans experiments. S.L.A. and R.A.M. did in vitro and cell-culture experiments. I.S. performed all whole fly experiments. K.M. generated fosmid reporters and imaged transgenic worms. T.R.B. and J.W. performed bioinformatics analyses. L.C., C.A. and S.L.A. wrote the paper and prepared the figures.

Corresponding authors

Correspondence to Stefan L Ameres or Luisa Cochella.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Ath-HEN1 methylates animal miRNAs but does not affect miRNA steady-state abundance in C. elegans and Drosophila.

(a) Confirmation of the efficiency of discriminatory 3´ ligation between methylated and non-methylated small RNA upon oxidation. Small RNAs from Drosophila S2 cells were ligated to a pre-adenylated, 3´ sequencing adapter, either before or after oxidation with periodate, and subjected to Northern hybridization analysis using probes against the indicated small RNAs. The endogenously methylated endo-siRNA esi-2.1 served as positive control, its ligation proceeds to completion even after oxidation. The unmethylated miRNA bantam-3p served as negative control, its ligation is abolished upon oxidation. Independent repeats = 1. (b) Expression of FLAG-Ath-HEN1 and endogenous Dme-Hen1 was assessed by Western blot analysis of lysate prepared from wild-type (S2WT) S2 cells or S2 cells depleted of endogenous Dme-Hen1 by CRISPR/Cas9 genome engineering (S2-hen1KO). If indicated, wild-type (WT) or catalytic mutant (CM) Ath-HEN1 was stably expressed. Actin served as loading control. Independent repeats = 2. (c) FLAG-tagged Ath-HEN1 was immunopurified upon expression in Drosophila S2 cells and incubated with 5´ 32P radiolabeled Drosophila let-7 (21 nt) or miR-34 (24 nt) in a single- (left panel) or double-stranded (right panel) conformation. Incubation with buffer served as negative control (Contr.). MicroRNA methylation was assessed by β-elimination and high-resolution polyacrylamide gel electrophoresis. Signal corresponding to methylated (mod.) or unmethylated (unmod.) miRNAs is indicated. Independent repeats = 3. (d) Ath-HEN1 methylates endogenous miRNAs in animal cells. Ath-HEN1 driven by a constitutive Actin5C (Act5C) promoter was expressed in Drosophila S2 cells depleted of endogenous Dme-Hen1 by CRISPR-Cas9 genome editing (S2-hen1KO) and methylation status of endogenous miRNAs or the endogenous siRNA esi-2.1 was assessed by β-elimination and high-resolution Northern hybridization using probes for the indicated miRNAs. Wild-type S2 cells are shown as control. Signal corresponding to methylated (mod.) or unmethylated (unmod.) miRNAs is indicated. 2SrRNA served as control for loading and β-elimination. Independent repeats = 2. (e) Expression of FLAG-MYC-Ath-HEN1 from a UAST or UASP transgenic construct driven by Act5C-Gal4 (Act5C-Gal4; UAST-Ath-HEN1 or Act5C-Gal4; UASP-Ath-HEN1) was assessed by anti-FLAG Western blotting. Lysate was prepared from whole adult 2-5-day old female wild-type flies (w1118) or flies depleted of endogenous Dm-Hen1 by CRISPR/Cas9 genome editing (w1118; hen1m1-6) carrying the indicated transgenes. Actin served as loading control. Independent repeats = 2. (f) Ath-HEN1 methylates animal miRNAs in vivo in Drosophila. Total RNA was extracted from whole adult 2-5-day old female wild-type flies (w1118) or flies depleted of endogenous Dm-Hen1 by CRISPR/Cas9 genome editing (w1118; hen1c4-6). If indicated, flies ubiquitously express Ath-HEN1 from a UAST or UASP transgenic construct driven by Act5C-Gal4 (Act5C-Gal4; UAST-Ath-HEN1 or Act5C-Gal4; UASP-Ath-HEN1). Total RNA was subjected to b-elimination and Northern hybridization using probes against the indicated miRNAs. 2SrRNA served as control for loading and b-elimination. Independent repeats = 2. (g) Expression of MYC-Ath-HEN1 under the rps-5prom in L1-stage worms was assessed by anti-MYC Western blotting. Two independent isolates from the transgene integration procedure were tested and further used for sequencing experiments. N2 animals that do not express MYC-Ath-HEN1 served as a negative control. Gamma tubulin staining was used as loading control. Independent repeats = 2. (h) Ath-HEN1 methylates animal miRNAs in vivo in C. elegans. Total RNA extracted from wild type or transgenic rps-5prom::Ath-HEN1 L1-stage worms was subjected to b-elimination and Northern hybridization using probes against the indicated miRNAs. tRNAGly served as control for loading and b-elimination. Independent repeats = 2. (i, j) Methylation does not affect miRNA relative abundance in S2 cells or worms. Comparison of the relative abundances of all expressed miRNAs in S2-hen1KO cells stably expressing wild type (WT) or catalytic-mutant (CM) Ath-HEN1 (i) or in wt, non-transgenic L1 worms versus animals expressing rps-5prom::Ath-HEN1 (j). Spearman correlation coefficients and associated p-Values are indicated. Independent repeats = 1 for i, 2 for j. For full scans of all blots in this figure see Suppl. Fig. 9 and 10.

Supplementary Figure 2 Signal and background constraints for mime-seq.

(a) Schematic of the domain structure of plant and animal HEN1. The Arabidopsis enzyme has two dsRNA binding domains that define its specificity for short duplexes. The animal methyltransferases, despite similar enzymatic activity, do not bind or act on dsRNA and in fact do not seem to share a common ancestor with Ath-HEN1. (b) Pie charts showing the proportion of miRNAs and other small RNAs sequenced before and after oxidation of RNA obtained from wild-type, non-trangenic C. elegans L1 larvae. While miRNAs are strongly depleted, the libraries generated after oxidation still contained 5-10% of reads corresponding to miRNAs, mostly representing 26 miRNAs (Suppl. Table 6). Given their precursor structure and association with other Argonautes, and the fact that they are depleted upon oxidation in henn-1(0) background (Suppl. Tables 3, 4, 5), these are endogenous targets of Cel-HENN-1 (see discussion and Suppl. Table 6). (c) Duplex structures of two of the miRNAs that are endogenously methylated by Cel-HENN-1. (d) Comparison of the relative abundances of all expressed mature miRNAs in Drosophila S2 cells depleted of endogenous Dme-Hen1 by CRISPR-Cas9 genome editing (S2-hen1KO) and expressing Ath-HEN1, before and after oxidation treatment. Spearman correlation coefficient and associated p-Value are shown. Independent repeats = 1. (e) Depletion of endogenous RNA methyltransferase HENN-1 does not affect miRNA relative abundance in worms. Comparison of the relative abundances of all confidently detected (>10 cpm in the average of all untreated samples) mature miR strands in wt, non-transgenic L1 worms versus animals deficient for endogenous HENN-1. Spearman correlation coefficient and associated p-Value are shown. Independent repeats = 1. (f) Comparison of the relative abundances of all confidently detected (>10 cpm in the average of all untreated samples) mature miR strands in henn-1(0) worm L1s expressing rps-5prom::Ath-HEN1, before and after oxidation treatment. Spearman correlation coefficient and associated p-Value are shown. In blue are miRNAs that are significantly depleted after oxidation (p-Value <0.001) and were not detected as enriched in any other profiling experiment in this study (see text and Supplementary Table 2). Independent repeats = 2. (g) Volcano plot of the data in (f) showing the respective p-Values for all fold-changes. Independent repeats = 2.

Supplementary Figure 3 Periodate-treatment enables selective cloning of animal miR and miR* species upon restrictive Ath-HEN1-directed methylation.

(a) Volcano plots show fold-change and associated p-Values (determined by DESeq2, see methods) for the indicated number (n) of abundantly expressed fly (red) or mouse (black) miRNAs in small RNA libraries generated as described in Fig. 2a from the indicated input ratios. True-positive miRNA-recovery rate (TPR) was determined from the number of abundantly expressed fly miRNAs detected in the respective libraries above cutoff (log2 fold-change >1; p-Value<10−3) relative to the 64 fly miRNAs recovered at a read depth of >10 cpm (na=64) in untreated 1:1 input libraries. False-positive miRNA-recovery rate (FPR) was determined from the number of mouse miRNAs above cutoff (log2 fold-change >1; p-Value<10−3). (b) As described in Fig. 2a, total RNA derived from Drosophila melanogaster (dme) S2hen1 cells expressing Ath-HEN1 was mixed with total RNA from mouse (mmu) embryonic stem cells at the indicated ratios (input ratio), followed by small RNA cloning before (-ox) and after (+ox) oxidation by periodate-treatment. This experiment was conducted in triplicate. Here we focus on the fraction of miR* strands mapping to annotated mouse (mmu miR) or fly miRNAs (dme miR). The number of abundantly expressed fly miR*s (>100 cpm in untreated 1:1 input ratio libraries) still detected after the respective dilutions at a read depth of >100 cpm is indicated in parenthesis. (c) Volcano plots show fold-change and associated p-Values (determined by DESeq2, see methods) for the indicated number (n) of miR* strands of abundantly expressed fly (blue) or mouse (black) miRNAs in small RNA libraries generated as described in Fig. 2a from the indicated input ratios. True-positive miRNA-recovery rate (TPR) was determined from the number of abundantly expressed fly miR* species detected in the respective libraries above cutoff (log2fold-change >1; p-Value<10−3) relative to the expected 64 fly miR* partner strands of miRs recovered at a read depth of >10 cpm (na=64) in untreated 1:1 input libraries. False-positive miRNA-recovery rate (FPR) was determined from the number of mouse miR* species above cutoff (log2 fold-change >1; p-Value<10−3).

Supplementary Figure 4 Ath-HEN1-directed methylation followed by oxidation-dependent cloning preserves the relative quantity of miR and miR* species.

Relative abundance of the indicated number of fly miR (red) and miR* (blue) species in the indicated small RNA libraries generated as described in Fig. 2a. Only miR and miR* strands that were significantly enriched (log2 fold-change > 1; p-Value<10−3) upon oxidation in the respective input dilution, as determined in Fig. 2b and Suppl. Fig. 3a and c, are shown. Spearman correlation coefficient (rs) and associated p-Value were determined separately for miR (red) and miR* (blue) species. Mean±stdev of three independent experiments are shown.

Supplementary Figure 5 Pan-neuronal mime-seq is highly reproducible and robust to Ath-HEN1 dose.

(a) Western blot analysis of the expression of MYC-Ath-HEN1 driven under three different pan-neuronal promoters in C. elegans (biological duplicates are shown). Tubulin detection was used for loading control. Non-transgenic, N2 animals were used for negative control. For full scans of the blots see Suppl. Fig. 10. Independent repeats = 2. (b, d, f) Comparison of the relative abundances of all confidently detected (>10 cpm in the average of all untreated samples) mature miR strands in henn-1(0) worm L1s expressing Ath-HEN1 under three different pan-neuronal drivers, before and after oxidation treatment, shows that some miRNAs are enriched while others are depleted by the oxidation treatment. (c, e, g) Comparison of the relative abundances of all confidently detected (>10 cpm in the average of all untreated samples) miRNAs for each pair of biological replicates, for both treated and untreated samples, in all three pan-neuronal experiments. Spearman correlation coefficients and their significance are shown. All experiments in this figure were performed on two biologically independent samples.

Supplementary Figure 6 Methylated miR* enrichment supports neuronal miRNA assignments.

For each pan-neuronal mime-seq experiment, we analyzed the change in relative abundance after periodate-mediated oxidation of all miR strands detected above an average of 10 cpm in all the untreated samples, as well as of all miR* strands corresponding to the detected miRs, regardless of their abundance. The correlations between the enrichment of both species is plotted for each experiment and the Pearson correlation coefficients (rp) and associated two-tailed p-Values (calculated in Prism7) are shown. The Venn diagrams display how many miR strands were significantly enriched (log2 fold-change > 1; p-Value<10−3) upon oxidation in the respective experiment and from their respective miR* strands, how many were also enriched (log2 fold-change > 1; p-Value<0.0013) and expressed above 10 cpm. The majority of miR* strands that do not support miR enrichment are expressed below 10 cpm in the unoxidized samples. Experiments in this figure were performed on two biologically independent samples.

Supplementary Figure 7 Mime-seq reveals the miRNomes of C. elegans tissues of diverse composition and size.

(a) Western blot analysis of the expression of MYC-Ath-HEN1 driven under different tissue-specific promoters in C. elegans (biological duplicates are shown). Tubulin detection was used for loading control. Non-transgenic, N2 animals were used for negative control. For full scans of the blots see Suppl. Fig. 10. Independent repeats = 2 for elt-2 and myo-2, 1 for myo-3. (b, d, f) Comparison of the relative abundances of all confidently detected (>10 cpm in the average of all untreated samples) mature miR strands in henn-1(0) worm L1s expressing Ath-HEN1 under three different tissue-specific drivers, before and after oxidation treatment, shows that some miRNAs are enriched while others are depleted by the oxidation treatment. (c, e, g) Comparison of the relative abundances of all confidently detected (>10 cpm in the average of all untreated samples) mature miR strands for each pair of biological replicates, for both treated and untreated samples, in all three tissue-specific experiments. Spearman correlation coefficients and their significance are shown. All experiments in this figure were performed on two biologically independent samples.

Supplementary Figure 8 Mime-seq can be performed in wild-type background in worms and flies.

(a) Expression of Ath-HEN1 in the ASE neurons was monitored by joint expression of mCherry-H2B using a T2A peptide. At least 10 animals were analyzed from each of two independent transgenic lines. Scale bar = 10 μm. (b) Comparison of the relative abundances of all confidently detected (>10 cpm in the average of all untreated samples) mature miR strands in henn-1(0) worm L1s expressing Ath-HEN1 under the ASE-specific promoter, before and after oxidation treatment. Reproducibility of the two biological replicates, for both treated and untreated conditions, are shown. Spearman correlation coefficients and their significance are shown. (c) Same as in (b) but with animals carrying wild-type (wt) endogenous henn-1. Experiments in this figure were performed on two biologically independent samples. (d) Venn diagram shows the overlap between the significantly enriched miRNAs (log2 Fold Change>1, p-Value<0.001 for henn-1(tm4477) or <0.01 for wt) in each of the two backgrounds. (e) Muscle mime-seq results from C. elegans from both henn-1(0) and wild-type backgrounds are shown as volcano plots. These show miRNAs enriched or depleted following oxidation treatment, and their associated p-Values. The wt enrichments shown are after subtraction of the background enrichment in non-transgenic, wt animals to account for endogenous Cel-HENN-1-mediated methylation. The Venn diagram shows the overlap between the significantly enriched miRNAs (log2 fold-change>1, p-Value<0.001 for henn-1(0) or <0.01 for wt) in each of the two backgrounds. Experiments in this figure were performed on two biologically independent samples. (f) Muscle mime-seq results from Drosophila using all muscle or flight-muscle-specific drivers are shown as volcano plots. These show miRNAs enriched or depleted following oxidation treatment, and their associated p-Values. The Venn diagram shows the overlap between the miRNAs enriched >2-fold in each of the two experiments. Experiments in this figure were performed in single replicates.

Supplementary Figure 9 Source images for Drosophila experiments.

Full scans of Northern hybridization and Western blots associated with (a) Suppl. Fig. 1c, (b) Suppl. Fig. 1a, (c) Suppl. Fig. 1b, (d) Suppl. Fig. 1d (black and red dotted box), (e) Suppl. Fig. 1e, (f) Fig. 1b (black dotted box) and Suppl. Fig. 1f (black and red dotted box). Marker lane is indicated (M).

Supplementary Figure 10 Source images for C. elegans experiments.

Full scans of Northern hybridization and Western blots associated with (a) Fig. 1c (black dotted box) and Suppl. Fig. 1h (red dotted box), (b) Suppl. Fig. 5a (red dotted box), (c) Suppl. Fig. 7a (long red dotted box) and Suppl. Fig. 1g (short red dotted box). Marker lane is indicated (M).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Tables 7–8 (PDF 2554 kb)

Life Sciences Reporting Summary (PDF 166 kb)

Supplementary Protocol

MicroRNome by methylation-dependent sequencing (mime-seq) (PDF 6917 kb)

Supplementary Table 1

Synthetic dilution experiment to determine thesensitivity of mime-seq (XLSX 1373 kb)

Supplementary Table 2

A few miRNAs are not efficiently methylated by AthHEN1 (XLSX 58 kb)

Supplementary Table 3

Pan-neuronal miRNAs from C. elegans revealed by mime-seq and comparison with transcriptional reporters (XLSX 111 kb)

Supplementary Table 4

Tissue-specific miRNAs from C. elegans revealed by mime-seq (XLSX 112 kb)

Supplementary Table 5

The miRNome of a single neuron class in C. elegans revealed by mime-seq in wild-type and henn-1(0) backgrounds (XLSX 164 kb)

Supplementary Table 6

miRNAs endogenously methylated by Cel-HENN-1 (XLSX 68 kb)

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Alberti, C., Manzenreither, R., Sowemimo, I. et al. Cell-type specific sequencing of microRNAs from complex animal tissues. Nat Methods 15, 283–289 (2018). https://doi.org/10.1038/nmeth.4610

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