Journal: Nature Communications

Article Title: Interplay of two small RNAs fine-tunes hierarchical flagella gene expression in Campylobacter jejuni

doi: 10.1038/s41467-024-48986-8

Figure Lengend Snippet: a Northern blot analysis of total RNA from C. jejuni WT and ribonuclease (RNase) deletion mutant strains in exponential phase. Colored triangles: prominent CJnc230 transcripts determined by termination site sequencing (term-seq) and/or primer extension. The flgE mRNA was detected with CSO-5136 (binding the coding sequence (CDS)), CJnc230 sRNA was probed with CSO-0537 (middle of the sRNA) or CSO-4297 (3’-extended versions, binding ~70 nt downstream of the annotated 3’ end ). RnpB RNA (CSO-0497) served as a loading control. Oligonucleotide binding positions are indicated in ( c ). b Primer extension analysis of CJnc230 5’ ends from C. jejuni WT and RNase deletion mutant strains in exponential phase with the CJnc230 probe used for northern blots (CSO-0537) binding in the middle of the sRNA. A sequencing ladder generated with this probe is partially indicated on the left. Black triangle: CJnc230 5’ end in the WT (C residue in bold on the left). Gray triangle: alternative 5’ end 28 nt upstream in Δ rnc (T residue in bold). c Term-seq and total RNA-seq represented by relative cDNA read counts at the CJnc230 locus in C. jejuni WT and nuclease deletion mutants grown to exponential phase. One representative biological replicate each is shown. For term-seq libraries, coverage for the last base of each read was plotted (3’nt), while total RNA-seq tracks depict full read coverage. Bent arrow: metB transcriptional start site (TSS) . Dashed arrows: prominent CJnc230 3’ ends (T 1 , T 2 , and T ) with resulting sRNA lengths in nucleotides and RNA 3’ end downstream of flgE (T flgE ). Oligonucleotide binding positions used for northern blot in ( a ) are indicated. d Reverse transcription-polymerase chain reaction (RT-PCR) analysis of total RNA from C. jejuni WT, Δ rnc , and Δ rny harvested at exponential growth phase. ( Left ) The flgE -CJnc230- metB locus. Arrows below: RT-PCR primers. Bent arrows: TSSs & promoter motifs , . ( Right ) Agarose gels of RT-PCR reactions performed in the presence (+) or absence (-) of reverse transcriptase (RT). Reactions with genomic DNA (gDNA) of WT or water (H 2 O) served as positive and negative controls, respectively. e Model of CJnc230 processing and maturation by three ribonucleases. RNase III potentially cleaves a predicted stem-loop in the flgE -CJnc230 transcript (Supplementary Fig. ), while RNase Y and PNPase are involved in maturation of the sRNA 3’ end (full-length CJnc230, 98 nt, colored in black) or in degradation of precursor transcripts. Data in ( a – d ) are representative results of at least two independent experiments. Source data are provided as a Source Data File.

Article Snippet: The RNA was dissolved in 11.5 μl H 2 O and 8.5 μl of a mix containing 2 μl 10 x T4 RNA ligase buffer (NEB), 3 μl DMSO, 2 μl ATP (1 mM final concentration; NEB), 1 μl T4 RNA ligase 1 (10 U; NEB), and 0.5 μl Ribonuclease Inhibitor (10 U; Molox) was added.

Techniques: Northern Blot, Mutagenesis, Sequencing, Binding Assay, Control, Generated, Residue, RNA Sequencing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction