Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

Abstract

The bacterial protein Hfq participates in the regulation of translation by small noncoding RNAs (sRNAs). Several mechanisms have been proposed to explain the role of Hfq in the regulation by sRNAs binding to the 5 ′ -untranslated mRNA regions. However, it remains unknown how Hfq affects those sRNAs that target the coding sequence. Here, the contribution of Hfq to the annealing of three sRNAs, RybB, SdsR, and MicC, to the coding sequence of Salmonella ompD mRNA was investigated. Hfq bound to ompD mRNA with tight, subnanomolar affinity. Moreover, Hfq strongly accelerated the rates of annealing of RybB and MicC sRNAs to this mRNA, and it also had a small effect on the annealing of SdsR. The experiments using truncated RNAs revealed that the contributions of Hfq to the annealing of each sRNA were individually adjusted depending on the structures of interacting RNAs. In agreement with that, the mRNA structure probing revealed different structural contexts of each sRNA binding site. Additionally, the annealing of RybB and MicC sRNAs induced specific conformational changes in ompD mRNA consistent with local unfolding of mRNA secondary structure. Finally, the mutation analysis showed that the long AU-rich sequence in the 5 ′ -untranslated mRNA region served as an Hfq binding site essential for the annealing of sRNAs to the coding sequence. Overall, the data showed that the functional specificity of Hfq in the annealing of each sRNA to the ompD mRNA coding sequence was determined by the sequence and structure of the interacting RNAs.

The binding of Class II sRNA MgrR to two different sites on matchmaker protein Hfq enables efficient competition for Hfq and annealing to regulated mRNAs

“…1). These observations support the conclusion that even though the role of Hfq is to locally rearrange RNA structures, to do so it needs at least partially unstable structure regions available for its binding (Wroblewska and Olejniczak 2016). The sequence motifs involved in Hfq-dependent annealing of MgrR to eptB and ygdQ mRNAs are located upstream of the MgrR binding site in both mRNAs (Figs.…”

“…The homohexameric, ring-shaped Hfq serves as a platform for simultaneous binding of sRNAs and regulated mRNAs to facilitate their annealing (Updegrove et al 2016). Additionally, Hfq rearranges the structures of interacting RNAs to enable the pairing of complementary sequences (Soper and Woodson 2008;Soper et al 2011;Wroblewska and Olejniczak 2016). To simultaneously engage different RNAs, Hfq uses three RNA binding sites on the proximal and distal faces of its ring, and on the rim (Mikulecky et al 2004;Link et al 2009;Sauer et al 2012).…”

“…The proximal face of Hfq binds the 3 ′ -terminal oligouridine tails of these sRNAs (Otaka et al 2011;Sauer and Weichenrieder 2011;Ishikawa et al 2012;Morita et al 2017), while the positively charged patches on the rim of Hfq contact the adenosine and uridine-rich sequences present in the central or 5 ′ -terminal regions of sRNA molecules (Sauer et al 2012;Dimastrogiovanni et al 2014). The distal face of Hfq recognizes adenosine-rich motifs, including repeated ARN sequences, which are present in mRNAs regulated by these sRNAs (de Haseth and Uhlenbeck 1980;Mikulecky et al 2004;Soper and Woodson 2008;Link et al 2009;Soper et al 2011;Peng et al 2014a,b;Wroblewska and Olejniczak 2016;Chen and Gottesman 2017;Andrade et al 2018). Hence, the canonical sRNAs and their mRNA targets bind to the opposite faces of Hfq, which enables Hfq to promote their annealing.…”

ProQ/FinO‐domain proteins: another ubiquitous family of RNA matchmakers?

“…The F plasmid FinO, E. coli ProQ and N. meningitidis NMB1681 proteins increased the rates of RNA annealing and strand exchange in in vitro assays using FinP sRNA and traJ mRNA fragments (van Biesen and Frost, ; Arthur et al ., ; Chaulk et al ., ). This role is reminiscent of the Hfq protein (Soper et al ., ; Wroblewska and Olejniczak, ). On the other hand, S. enterica ProQ protein induced a strong reverse transcriptase stop signal in a toeprinting assay of hupA mRNA bound by RaiZ sRNA, which was interpreted as the stabilization of the complex by ProQ (Smirnov et al ., ).…”

Hfq‐dependent mRNA unfolding promotes sRNA ‐based inhibition of translation

“…Interaction with Hfq protects many RNAs from rapid degradation (Moll et al, 2003a;Holmqvist et al, 2010) and promotes the annealing of sRNAs and their mRNA targets (Møller et al, 2002;Zhang et al, 2002;Lease & Woodson, 2004;Fender et al, 2010;Wroblewska & Olejniczak, 2016). Several non-exclusive models suggest how Hfq can facilitate intermolecular base-pairing.…”