Feedback regulation of small RNA processing by the cleavage product

Abstract

Many bacterial small RNAs (sRNAs) are processed resulting in variants with roles potentially distinct from the primary sRNAs. In Enterobacteriaceae sRNA GlmZ activates expression of glmS by base-pairing when the levels of glucosamine-6-phosphate (GlcN6P) are low. GlmS synthesizes GlcN6P, which is required for cell envelope biosynthesis. When dispensable, GlmZ is cleaved by RNase E in the base-pairing sequence. Processing requires protein RapZ, which binds GlmZ and recruits RNase E by interaction. Cleavage is counteracted by the homologous sRNA GlmY, which accumulates upon GlcN6P scarcity and sequesters RapZ. Here, we report a novel role for a processed sRNA. We observed that processing of GlmZ is never complete in vivo . Even upon RapZ overproduction, a fraction of GlmZ remains full-length, while the 5ʹ cleavage product (GlmZ*) accumulates. GlmZ* retains all elements required for RapZ binding. Accordingly, GlmZ* can displace full-length GlmZ from RapZ and counteract processing in vitro . To mimic GlmZ* in vivo , sRNA chimeras were employed consisting of foreign 3ʹ ends including a terminator fused to the 3ʹ end of GlmZ*. In vitro , these chimeras perform indistinguishable from GlmZ*. Expression of the chimeras in vivo inhibited processing of endogenous GlmZ, causing moderate upregulation of GlmS synthesis. Hence, accumulation of GlmZ* prevents complete GlmZ turnover. This mechanism may serve to adjust a robust glmS basal expression level that is buffered against fluctuations in RapZ availability.

Small RNA ‐binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli

“…Moreover, the low glmY expression level in the ΔrapZ mutant remained unaffected by overproduction of the sRNAs. As deletion or overproduction of GlmY or GlmZ has no impact on RapZ levels (Durica‐Mitic & Görke, ), these results suggested that GlmY and GlmZ counteract activation of QseE/QseF by RapZ.…”

“…Total RNA was extracted and analyzed by Northern blotting as described recently (Durica‐Mitic & Görke, ). For RNA half‐life determinations, 500 μg/ml rifampicin was added to the cultures when reaching OD 600 = 1.0 and aliquots were harvested at indicated times by pelleting and freezing in liquid nitrogen.…”

Adaptor protein RapZ activates endoribonuclease RNase E by protein–protein interaction to cleave a small regulatory RNA

“…For confirmation, we made use of a previously established RNase E cleavage assay (Göpel et al 2013(Göpel et al , 2016Durica-Mitic and Görke 2019) to analyze activity of the RapZ CTD in vitro. In this assay, correct cleavage of radiolabeled GlmZ yielding the 155 nt long 5 ′ cleavage product (designated GlmZ * ) only occurs in the presence of both RapZ and Rne NTD (Fig.…”

RNA and DNA G-quadruplexes bind to human dicer and inhibit its activity

“…Accordingly, we hypothesize that short RNAs adopting G-quadruplex structures may sequester Dicer in vivo. Such a mechanism could be a part of autoregulatory loops, in which Dicer-processed guanine-rich miRNAs, in the form of G-quadruplexes, act in a negative feedback regulation of Dicer [ 17 , 41 ]. The sequestration of Dicer by RNA G-quadruplexes may control the pool of Dicer available for the biogenesis of miRNA.…”