Extended Data Fig. 1: miRNA from plants and mammals are pseudouridylated.
From: Pseudouridine guides germline small RNA transport and epigenetic inheritance
(a) Immunoprecipitation with anti-Ψ antibody enriches for Ψ-containing RNAs produced through in vitro transcription (IVT) as determined by mass spectrometry (n = 3 biological replicates, error bars ± SD). (b) Immuno-dot blot of 100 ng total input RNA or 100 ng RNA immunoprecipitated with anti-Ψ antibody or control immunoglobulin G (IgG). 100 ng of 100% Ψ IVT RNA was used as control. (c) Volcano plot of miRNAs enriched by small RNA immunoprecipitation using a Ψ-specific antibody (Ψ-IP) in mouse fibroblast NIH/3T3 cells compared to input fractions. Significantly enriched and depleted miRNAs are highlighted in blue (padj<0.01; DESeq2, Benjamini and Hochberg correction for multiple comparisons; n = 3 biological replicates). (d) Validation of enriched miRNAs using qRT-PCR. (e) Ψ-enrichment of miRNAs affected by PUS1 knockdown (KD) 3 days and 6 days after PUS1 shRNA transfection of NIH/3T3 cells. (f) Arabidopsis AGO proteins and Dyskerin subunit NHP2 were fused at the N-terminus to N- and C- terminally split YFP, respectively. Positive interaction is shown by YFP signal, with 35S::RFP as an expression control. (g) Ψ-enrichment of precursor pri-miRNAs in Arabidopsis flower buds plotted against pri-miRNAs bound to NHP2 (blue) and detected by RIP-seq in 2 or more replicates (orange). (h) Violin plot showing significant difference between NHP2-enriched and non-enriched pri-miRNA in Ψ enrichment of the mature miRNA by Ψ -IP (Log2 IP/IgG; numbers below violins indicate number of miRNA analyzed;one-way ANOVA; **p < 0.01).
