Evaluation of 5′-End Phosphorylation for Small RNA Stability and Target Regulation In Vivo

Alexandra Schilder, Yvonne Göpel, Muna Ayesha Khan, Boris Görke (Korresp. Autor*in)

Veröffentlichungen: Beitrag in BuchBeitrag in Buch/SammelbandPeer Reviewed

Abstract

Bacterial small RNAs (sRNAs) can be equipped at the 5′ end with triphosphate (5′PPP) or monophosphate (5′P) groups, depending on whether they are primary transcripts, undergo dephosphorylation or originate via processing. Often, 5′ groups hallmark RNAs for rapid decay, but whether this also applies to sRNAs is little explored. Moreover, the sRNA 5′P group could activate endoribonuclease RNase E to cleave the base-paired target RNA, but a tool for investigation in vivo was lacking. Here, we describe a two-plasmid system suitable for the generation of 5′ monophosphorylated RNAs on demand inside the cell. The sRNA gene of interest is fused to the 3′ end of a fragment of sRNA GlmZ and transcribed from a plasmid in an IPTG-inducible manner. The fusion RNA gets cleaved upon arabinose-controlled expression of rapZ, provided on a compatible plasmid. Adaptor protein RapZ binds the GlmZ aptamer and directs RNase E to release the sRNA of choice with 5′P ends. An isogenic plasmid generating the same sRNA with a 5′PPP end allows for direct comparison. The fates of the sRNA variants and target RNA(s) are monitored by Northern blotting. This tool is applicable to E. coli and likely other enteric bacteria.

OriginalspracheEnglisch
TitelMethods in Molecular Biology
Herausgeber (Verlag)Humana Press, Inc.
Seiten255-272
Seitenumfang18
DOIs
PublikationsstatusVeröffentlicht - 2024

Publikationsreihe

ReiheMethods in Molecular Biology
Band2741
ISSN1064-3745

ÖFOS 2012

  • 106023 Molekularbiologie
  • 106022 Mikrobiologie

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