New aspects of RNA-based regulation by Hfq and its partner sRNAs

Abstract

Hfq, an RNA chaperone, promotes the pairing of small RNAs (sRNAs) to target mRNAs, mediating post-transcriptional regulation of mRNA stability and translation. This regulation contributes to bacterial adaptation during stress and pathogenesis. Recent advances in sequencing techniques demonstrate the presence of sRNAs encoded not only in intergenic regions but also from the 3' and 5' UTRs of mRNAs, expanding sRNA regulatory networks. Additional layers of regulation by Hfq and its associated RNAs continue to be found. Newly identified RNA sponges modulate the activity of some sRNAs. A subset of sRNAs are proving to be bifunctional, able to pair with targets and also encoding small ORFs or binding other RNA binding proteins, such as CsrA. In addition, there are accumulating examples of Hfq inhibiting mRNA translation in the absence of sRNAs.

Figure 1:. Mechanisms of Hfq-dependent regulation

In general, binding of Hfq, with or without other partners (sRNAs in this figure) can change RNA folding and allow or block access of other RNA binding factors, therefore changing the fate of the mRNA. A. sRNA Regulation of Translation Initiation and/or mRNA Decay. Negative Regulation: In most cases, negative regulation is via sRNA pairing with targets, as shown here. This can lead to inhibition of translation (left panel), recruitment of RNases (right panel), or both (center panel). Hfq helps to stabilize and recruit the sRNA, and may, in some cases, help to recruit ribonucleases. Positive Regulation: sRNAs and Hfq binding can collaborate to change RNA folding, remodeling an inhibitory hairpin, for instance, to allow ribosome access (left and center panels), or blocking access of a ribonuclease such as RNase E (right panel), thus stabilizing an mRNA. B. Regulation of Rho-Dependent Transcription Termination Rho (purple hexamers) terminates transcription by first accessing naked RNA at a rut site (dotted yellow portion of mRNA), and then traveling along the elongating RNA to release the RNA from RNA polymerase (blue oval) (reviewed in [43]). Thus sRNAs that affect access to the rut site can regulate the ability of Rho to act. Negative Regulation: Promoting Rho-Dependent Termination: In at least one case, sRNA pairing to a target blocks ribosome entry, allowing Rho to access the naked RNA and leading to Rho-dependent termination within the ORF, downstream of the pairing site [44]. Positive Regulation: Blocking Rho-Dependent Termination: sRNA binding may also block access of Rho to RNA, therefore allowing transcription of downstream genes. In the case examined, this positive regulation collaborates with remodeling of the 5’ UTR to allow both increased transcription and translation [45].

Figure 2:. sRNA Synthesis

A. Many sRNAs are transcribed from intergenic regions as free-standing transcripts [11] [12] (Spot 42, reviewed in [55], is shown as an example); their abundance in the cell is primarily regulated at the level of transcription, and this is frequently highly regulated. B. sRNAs can also be synthesized from processing of mRNAs (as for CpxP; [19]); in this case, regulatory signals for the mRNA promoter will also govern synthesis of the sRNA. C. sRNA promoters may also be embedded within mRNA coding regions (as for MicL here; [18]). In this case, regulation of the upstream gene can be independent of transcription of the overlapping mRNA. The promoter for MicL is a sigma E-dependent promoter [18]; further processing takes place to create the final sRNA. D. Evidence has also accumulated for sRNAs arising from the 5’ UTR of mRNAs [15]. In the case shown, it is not clear that RirA is an Hfq-dependent sRNA (RirA) [20].

Figure 3:. RNA binding proteins and regulation by titrating sRNAs

RNA binding proteins can be removed from their regulatory sites on target mRNAs by competing RNAs that also carry the regulatory sites. These titrating RNAs may be the major regulatory input affecting translational regulatory proteins. A. Titration of CsrA: CsrA is a major regulatory protein that acts by binding to targets, frequently blocking ribosome access (reviewed in [32]). All bacteria that contain CsrA or its homologs also encode titrating sRNAs such as CsrB and CsrC, which each contain multiple CsrA binding sites. Recent work demonstrates that some Hfq-binding sRNAs that act by pairing, such as McaS, also contain CsrA binding sites and thus can titrate CsrA as well [30,33]. Different regulatory signals affect synthesis of different titrating sRNAs. B. In Pseudomonads, Hfq acts in concert with another RNA binding protein, Crc, to carry out sRNA-independent catabolite repression at multiple sites. The CrcZ regulatory RNA, when synthesized, acts to remove Hfq and Crc from targets, allowing expression of genes such as amiE (shown here) [52]. The expression of CrcZ is dependent upon a response regulator, CbrB, which responds to the status of carbon sources to set the hierarchy of carbon utilization [51].

Figure 3:. RNA binding proteins and regulation by titrating sRNAs

RNA binding proteins can be removed from their regulatory sites on target mRNAs by competing RNAs that also carry the regulatory sites. These titrating RNAs may be the major regulatory input affecting translational regulatory proteins. A. Titration of CsrA: CsrA is a major regulatory protein that acts by binding to targets, frequently blocking ribosome access (reviewed in [32]). All bacteria that contain CsrA or its homologs also encode titrating sRNAs such as CsrB and CsrC, which each contain multiple CsrA binding sites. Recent work demonstrates that some Hfq-binding sRNAs that act by pairing, such as McaS, also contain CsrA binding sites and thus can titrate CsrA as well [30,33]. Different regulatory signals affect synthesis of different titrating sRNAs. B. In Pseudomonads, Hfq acts in concert with another RNA binding protein, Crc, to carry out sRNA-independent catabolite repression at multiple sites. The CrcZ regulatory RNA, when synthesized, acts to remove Hfq and Crc from targets, allowing expression of genes such as amiE (shown here) [52]. The expression of CrcZ is dependent upon a response regulator, CbrB, which responds to the status of carbon sources to set the hierarchy of carbon utilization [51].

Figure 4:. Hfq Regulation in the absence of sRNAs

Hfq is able to bind RNA to repress mRNA translation. In some cases, this binding is close to or at the ribosome binding site, directly blocking ribosome access [47]. In at least one other case, binding is upstream of the ribosome binding site but leads to remodeling of the RNA to block ribosome access [48].