6) sRNAs involved in chromosome structure: the case of HU andREP RNA
Since the 1970s, researchers have acknowledged that RNA molecules participate in nucleoid condensation through RNA-DNA interactions (Hecht and Pettijohn, 1976). It has been hypothesised that nascent RNAs, or a distinctive class of unknown RNAs, stabilise the nucleoid. Given the importance of RNA molecules, along with HU and other nucleoid proteins, in maintaining chromosome structure, Macvanin et al. used a ribonomic approach to identify RNAs bound to HU (Macvanin et al. , 2012). In addition to tRNA and rRNA, HU binds to 11 mRNAs encoding membrane-associated proteins, four sRNAs (SsrS, SsrA, Ffs, and RnpB) and 10 novel non-coding RNAs (nc1 to nc10). Some of the sRNA targets were found to be transcribed from regions containing repetitive palindromic extragenic DNA elements, also known as REPs . A subsequent study by the same group confirmed the presence of 30 sRNAs transcribed from REP elements, which were found to be associated with the nucleoïd (Qianet al. , 2015). REP elements were first reported in enterobacterial genomes 30 years ago (Gilson et al. , 1991) and consist of single palindromes separated by linkers. The E. coli genome has nearly 350 annotated REP elements, more than 50% of which are transcribed (Qian et al. , 2015). Therefore, transcripts involved in chromosome condensation are referred to as nucleoid-associated ncRNAs or naRNAs. Further research on the relationship between RNA and chromosome condensation has mainly focused on REP325 , which corresponds to nc5 (Qianet al. , 2015). REP325 consists of six highly homologous repeats composed of palindromic cruciform motifs, called Y and Z2 which generate motifs separated by five spacers with the same DNA sequence (Figure 5a ). REP325 is transcribed into six RNAs, naRNA1 to naRNA6. Each naRNA consists of two potential hairpins (corresponding to motifs Y and Z2 ) connected by a short linker (Figure 5a ). Interestingly, a strain withoutREP325 shows a greater degree of nucleoid decompaction that can only be restored by the presence of naRNA4. In addition, a chromosome conformation capture (3C) targeting REP segments revealed a physical proximity between different REP elements. This physical connection depends on the presence of both HU and REP325 RNA. However, other NAPs and RNAs may also be involved. Molecular analyses have shown that the effect of RNAs on DNA condensation requires the presence of two hairpins (Y andZ2 ) rather than a specific sequence (Qian, Zhurkin and Adhya, 2017), and that HU connects to naRNA4. The model suggests that naRNA4 uses its two bulged hairpins to form DNA-RNA complexes through potential secondary structure. This configuration is only possible when DNA is supercoiled and palindromic sequences are converted into cruciform structures, which aids with chromosome condensation (Figure 5b ). Cruciform DNA and RNA hairpins can bind to the HU protein, allowing the formation of DNA-naRNA4 complexes (Qian, Zhurkin and Adhya, 2017), and recent data suggest that HU must be able to bind both HU and the DNA in order to execute its function (Bettridge et al., 2019, unpublished data). However, it is worth noting that after facilitating the formation of the DNA-RNA complex, the HU protein dissociates from the complex, suggesting a chaperon-like role for HU (Qian, Zhurkin and Adhya, 2017).