Journal: bioRxiv

Article Title: Interplay of two small RNAs fine-tunes hierarchical flagellar gene expression in the foodborne pathogen Campylobacter jejuni

doi: 10.1101/2023.04.21.537696

Figure Lengend Snippet: (A) Northern blot analyses 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 sRNA) or CSO-4297 (3’-extended versions, binding ∼70 nt downstream of 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 using 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 cDNA coverages at the CJnc230 locus in C. jejuni WT and nuclease deletion mutants grown to exponential phase. One representative 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) from . Dashed arrows: prominent CJnc230 3’ ends (T 1 , T 2 , and T 3 ) 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 ( Fig. S3 ), 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.

Article Snippet: A sequencing ladder was generated using the DNA Cycle Sequencing kit (Jena Bioscience) according to the manufacturer’s recommendations with the CJnc230 sRNA region amplified from NCTC11168 WT (CSS-5295) gDNA using oligonucleotides CSO-3995 x 3993.

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