Nicholas De Lay

Bacterial Small RNA-based Negative Regulation: Hfq and its Accomplices

A large group of bacterial small regulatory RNAs (sRNAs) use the Hfq chaperone to mediate pairing with and regulation of mRNAs. Recent findings help to clarify how Hfq acts and highlight the role of the endonuclease RNase E and its associated proteins (the degradosome) in negative regulation by these sRNAs. sRNAs frequently uncouple transcription and translation by blocking ribosome access to the mRNA, allowing other proteins access to the mRNA. As more examples of sRNA-mediated regulation are studied, more variations on how Hfq, RNase E, and other proteins collaborate to bring about sRNA-based regulation are being found.

A small RNA that regulates motility and biofilm formation in response to changes in nutrient availability in Escherichia coli

In bacteria, many small regulatory RNAs (sRNAs) are induced in response to specific environmental signals or stresses and act by base‐pairing with mRNA targets to affect protein translation or mRNA stability. In Escherichia coli, the gene for the sRNA IS061/IsrA, here renamed McaS, was predicted to reside in an intergenic region between abgR, encoding a transcription regulator and ydaL, encoding a small MutS‐related protein. We show that McaS is a ∼ 95 nt transcript whose expression increases over growth, peaking in early‐to‐mid stationary phase, or when glucose is limiting. McaS uses three discrete single‐stranded regions to regulate mRNA targets involved in various aspects of biofilm formation. McaS represses csgD, the transcription regulator of curli biogenesis and activates flhD, the master transcription regulator of flagella synthesis leading to increased motility, a process not previously reported to be regulated by sRNAs. McaS also regulates pgaA, a porin required for the export of the polysaccharide poly β‐1,6‐N‐acetyl‐d‐glucosamine. Consequently, high levels of McaS result in increased biofilm formation while a strain lacking mcaS shows reduced biofilm formation. Based on our observations, we propose that, in response to limited nutrient availability, increasing levels of McaS modulate steps in the progression to a sessile lifestyle.

A complex network of small non‐coding RNAs regulate motility in Escherichia coli

Small Hfq‐dependent non‐coding regulatory RNAs (sRNAs) that alter mRNA stability and expression by pairing with target mRNAs have increasingly been shown to be important in influencing the behaviour of bacteria. In Escherichia coli, flhD and flhC, which encode the master regulator of flagellar synthesis, are co‐transcribed from a promoter that is regulated by multiple transcription factors that respond to different environmental cues. Here, we show that the 5′ untranslated region (5′ UTR) of the flhDC mRNA also serves as a hub to integrate additional environmental cues into the decision to make flagella. Four sRNAs, ArcZ, OmrA, OmrB and OxyS, negatively regulated and one sRNA, McaS, positively regulated motility and flhDC expression by base‐pairing with the 5′ UTR of this mRNA. Another sRNA, MicA, positively regulated motility independent of regulation of flhDC. Furthermore, we demonstrate that the regulation of motility by the ArcB/A two component system is in part due to its regulation of ArcZ. flhDC is the first mRNA that has been shown to be both positively and negatively regulated by direct pairing to sRNAs. Moreover, both positive regulation by McaS and negative regulation by ArcZ require the same binding site in the flhDC mRNA.

Alternative Pathways for Escherichia coli Biofilm Formation Revealed by sRNA Overproduction

Small regulatory RNAs have major roles in many regulatory circuits in Escherichia coli and other bacteria, including the transition from planktonic to biofilm growth. We tested Hfq‐dependent sRNAs in E. coli for their ability, when overproduced, to inhibit or stimulate biofilm formation, in two different growth media. We identify two mutually exclusive pathways for biofilm formation. In LB, PgaA, encoding an adhesion export protein, played a critical role; biofilm was independent of the general stress factor RpoS or CsgD, regulator of curli and other biofilm genes. The PgaA‐dependent pathway was stimulated upon overproduction of DsrA, via negative regulation of H‐NS, or of GadY, likely by titration of CsrA. In yeast extract casamino acids (YESCA) media, biofilm was dependent on RpoS and CsgD, but independent of PgaA; RpoS appears to indirectly negatively regulate the PgaA‐dependent pathway in YESCA medium. Deletions of most sRNAs had very little effect on biofilm, although deletion of hfq, encoding an RNA chaperone, was defective in both LB and YESCA. Deletion of ArcZ, a small RNA activator of RpoS, decreased biofilm in YESCA; only a portion of this defect could be bypassed by overproduction of RpoS. Overall, sRNAs highlight different pathways to biofilm formation.

The unmasking of ‘junk’ RNA reveals novel sRNAs: from processed RNA fragments to marooned riboswitches

While the notion that RNAs can function as regulators dates back to early molecular studies of gene regulation of the lac operon, it is only over the last decade that the ubiquity and diversity of regulatory RNAs are being realized. Advancements in high throughput sequencing and the adoption of these approaches to rapidly sequence genomes and transcriptomes and to examine gene expression and RNA binding protein specificity have revealed an ever-expanding RNA world. In this review, we focus on recent studies revealing that RNA fragments cleaved from larger coding or noncoding RNAs can have regulatory functions. Additionally, we discuss examples of riboswitches that function in trans as mRNA or protein-binding sRNAs, upending the traditional thinking that these are exclusively cis-acting elements.

Polynucleotide phosphorylase: Not merely an RNase but a pivotal post-transcriptional regulator

Almost 60 years ago, Severo Ochoa was awarded the Nobel Prize in Physiology or Medicine for his discovery of the enzymatic synthesis of RNA by polynucleotide phosphorylase (PNPase). Although this discovery provided an important tool for deciphering the genetic code, subsequent work revealed that the predominant function of PNPase in bacteria and eukaryotes is catalyzing the reverse reaction, i.e., the release of ribonucleotides from RNA. PNPase has a crucial role in RNA metabolism in bacteria and eukaryotes mainly through its roles in processing and degrading RNAs, but additional functions in RNA metabolism have recently been reported for this enzyme. Here, we discuss these established and noncanonical functions for PNPase and the possibility that the major impact of PNPase on cell physiology is through its unorthodox roles.