Gene autoregulation by 3’ UTR-derived bacterial small RNAs

Abstract

Negative feedback regulation, that is the ability of a gene to repress its own synthesis, is the most abundant regulatory motif known to biology. Frequently reported for transcriptional regulators, negative feedback control relies on binding of a transcription factor to its own promoter. Here, we report a novel mechanism for gene autoregulation in bacteria relying on small regulatory RNA (sRNA) and the major endoribonuclease, RNase E. TIER-seq analysis (transiently-inactivating-an-endoribonuclease-followed-by-RNA-seq) revealed ~25,000 RNase E-dependent cleavage sites in Vibrio cholerae, several of which resulted in the accumulation of stable sRNAs. Focusing on two examples, OppZ and CarZ, we discovered that these sRNAs are processed from the 3’ untranslated region (3’ UTR) of the oppABCDF and carAB operons, respectively, and base-pair with their own transcripts to inhibit translation. For OppZ, this process also triggers Rho-dependent transcription termination. Our data show that sRNAs from 3’ UTRs serve as autoregulatory elements allowing negative feedback control at the post-transcriptional level.

Bacterial 3′UTRs: A Useful Resource in Post-transcriptional Regulation

“…In addition, other RNA-seq based techniques that were envisioned for the identification of RNA-RNA and RNA-protein interactions and ribonuclease processing sites have also provided the scientific community with novel putative functional 3′UTR candidates. For example, the RIL-seq (RNA interaction by ligation and sequencing), RIP-seq (RNA immunoprecipitation followed by RNA sequencing), TIER-seq (transiently-inactivating-an-endoribonuclease-followed-by-RNA-seq) and CLASH (UV cross-linking, ligation, and sequencing of hybrids) methods have revealed dozens of sRNAs candidates originated from 3′UTRs of several enterobacterial species (Holmqvist et al, 2016 , 2018 ; Melamed et al, 2016 , 2019 ; Chao et al, 2017 ; Hoyos et al, 2020 ; Huber et al, 2020 ; Iosub et al, 2020 ). Combining these technologies with those dedicated to 5′ and 3′ end mapping (Sharma et al, 2010 ; Dar et al, 2016 ) would complete the bacterial transcriptomic landscapes and their RNA-RNA and RNA-protein network interactions, which are essential for understanding post-transcriptional regulatory mechanisms, including those involving 3′UTRs.…”

“…Besides, 3′UTR-derived sRNAs also participate in autoregulating the expression of the genes encoded in the same mRNAs that generate them. For example, the V. cholerae OppZ and CarZ sRNAs, which originate from the oppABCDF and carAB operons upon RNase E processing, respectively, base-pair with their own transcripts, leading to translation initiation inhibition and followed by Rho-dependent transcription termination (Hoyos et al, 2020 ). It is noteworthy that most of 3′UTR-derived sRNAs modulate the expression of proteins with functions related to the genes they originate from.…”

Small RNA Regulation of Virulence in Pathogenic Escherichia coli

“…However, the analysis used to identify these sRNAs searched for orphan Hfq-binding sites that were >100 nt from any annotated coding region. Recently, the 3’UTR of coding genes have emerged as a significant source of regulatory sRNAs, and have been found to regulate the outer membrane stress response, nitrate transport, and acetate and carbon metabolism ( Chao et al., 2012 ; Chao and Vogel, 2016 ; De Mets et al., 2019 ; Hoyos et al., 2020 ; Iosub et al., 2020b ; Wang et al., 2020 ). Taken together, this suggests that there may be many more pathotype-specific sRNAs than we currently appreciate, and it will be intriguing to investigate how these 3’UTR, 5’UTR, and recently identified CDS-encoded sRNAs may affect bacterial virulence.…”

“…Since their initial discovery from intergenic regions of genes however, a slew of novel biogenesis pathways and regulatory mechanisms for sRNAs have been uncovered. sRNAs have since been found to originate from 3’ UTRs ( Chao et al., 2012 ; Chao and Vogel, 2016 ; De Mets et al., 2019 ; Hoyos et al., 2020 ; Wang et al., 2020 ), 5’UTRs ( Vanderpool and Gottesman, 2004 ; Thomason et al., 2019 ), protein-coding transcripts ( Dar and Sorek, 2018 ), and pre-tRNAs ( Lalaouna et al., 2015 ), and can effect regulation of their targets by modulating Rho-dependent termination ( Sedlyarova et al., 2016 ; Silva et al., 2019 ; Hoyos et al., 2020 ), ribosome loading ( Jagodnik et al., 2017 ; Romilly et al., 2019 ) and sponging interactions ( Tree et al., 2014 ; Lalaouna et al., 2015 ; Miyakoshi et al., 2015 )…”

Suppression of Escherichia coli Growth Dynamics via RNAs Secreted by Competing Bacteria

“…Many cellular oligonucleotides are products of endonuclease cleavage (Davis and Waldor, 2007;Papenfort et al, 2009;Chao et al, 2017), and some short fragments of tRNAs are proven to be functional (Lalaouna et al, 2015a,b;Diebel et al, 2016;Swiatowy and Jagodzińśki, 2018). In recent years it has been also witnessed for both bacteria and higher organisms that 3 ′ -terminal regions of mRNAs can also be sources of short regulatory RNAs specifically processed by RNAse E (Eisenhardt et al, 2018;Miyakoshi et al, 2019;Hoyos et al, 2020;Wang et al, 2020). Thus, it is likely that the processing of many RNAs with well-established function plays a specific role in living cells by providing short oligonucleotides for diverse regulatory networks.…”