Journal: bioRxiv
Article Title: RNA-RNA Interactome Approaches Provide in vivo Evidence for a Critical Role of the Hfq Rim Face in sRNA-mRNA Pairing
doi: 10.1101/2025.06.26.660752
Figure Lengend Snippet: (A) Schematic overview of Hi-GRIL-seq and iRIL-seq workflows. WT (XL2001) and the hfq R16A mutant (FDM1050) were transformed with either an empty vector (pBAD24) or a T4 RNA Ligase I-expressing vector (pBAD24- t4rnl1 ). Biological duplicates of each strain were cultured in LB (Lennox) with ampicillin at 37°C until reaching an OD600 of ∼0.5, at which point arabinose was added to induce enzyme expression. Cells were then grown to early stationary phase (OD600 ∼1.0), harvested, and processed for either Hfq IP followed by RNA isolation (iRIL-seq) or direct RNA extraction for total RNA sequencing (Hi-GRIL-seq). sRNAs are shown in orange, mRNAs in black, tRNA in green and rRNA in blue. This figure was created in BioRender ( https://BioRender.com/jqqlsju ). (B) Circos plots illustrating RNA interactomes in WT and R16A Hfq with either pBAD24 or pBAD24- t4rnl1 , as detected by iRIL-seq. (C) Quantification of S-chimeras under different conditions (Supplementary Table S11). Table displaying the number of RNA pairs with significant chimeric fragments identified by iRIL-seq, with values shown for two biological replicates and their calculated mean. (D) Chimera levels of specific RNA pairs identified by RIL-seq (anti-Hfq) and iRIL-seq. The RNA pairs shown here include the six pairs analyzed in previous experiments as well as four additional pairs identified as having significant numbers of chimeras in the iRIL-seq R16A experiment. Chimera counts were extracted from Supplementary Table S6 (RIL-seq, anti-Hfq) and Supplementary Table S11 (iRIL-seq), using the same method described in . For each RNA pair, the values represent the mean from two biological replicates.
Article Snippet: The T4 RNA Ligase I-expressing plasmid pBAD24- t4rnl1 was synthesized by the GenScript company.
Techniques: Mutagenesis, Transformation Assay, Plasmid Preparation, Expressing, Cell Culture, Isolation, RNA Extraction, RNA Sequencing
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