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.
Highlights
The Hfq protein is involved in the regulation of translation by bacterial small RNAs (Updegrove et al 2016)
An (ARN)4 motif is important for Hfq binding and DsrA sRNA annealing to rpoS mRNA (Soper and Woodson 2008), an AU-rich sequence is involved in the regulation of chiP mRNA, while both ARN repeats and AU-rich regions serve in the regulation of csgD mRNA by different sRNAs (Schu et al 2015)
The Hfq-dependent acceleration of sRNA annealing to the mRNA untranslated region has been previously observed, for example, for OxyS annealing to fhlA mRNA (Zhang et al 2002; Salim and Feig 2010), Spot42 to galK mRNA (Moller et al 2002), and RNA-OUT to RNA-IN (Ross et al 2013)
Summary
The Hfq protein is involved in the regulation of translation by bacterial small RNAs (sRNAs) (Updegrove et al 2016) These noncoding RNAs recognize complementary sequences in their target mRNAs, and induce the activation or repression of translation (Waters and Storz 2009; Updegrove et al 2015). Hfq is an Sm-like protein, which has a shape of a homohexameric ring (Moller et al 2002; Zhang et al 2002). It binds uridine-rich sRNAs using its proximal face (Mikulecky et al 2004; Sauer and Weichenrieder 2011; Panja et al 2015) and the outer rim (Sauer et al 2012), and it binds mRNAs containing (ARN)n sequence motifs using its distal face (de Haseth and Uhlenbeck 1980; Link et al 2009). Recent studies have suggested that other modes of RNA interactions with Hfq are possible, both in Escherichia coli (Zhang et al 2013; Małecka et al 2015; Schu et al.2015) and in Gram-positive bacteria (Kovach et al 2014; Robinson et al 2014)
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