Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis depends on the ability to adjust to stresses encountered in a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. In spite of this, the understanding of M. tuberculosis RNA biology remains limited. Here we have used a DosR-associated sRNA as an example to investigate multiple aspects of mycobacterial RNA biology that are likely to apply to other M. tuberculosis sRNAs and mRNAs. We have found that accumulation of this particular sRNA is slow but robust as cells enter stationary phase. Using reporter gene assays, we find that the sRNA core promoter is activated by DosR, and we have renamed the sRNA DrrS for DosR Regulated sRNA. Moreover, we show that DrrS is transcribed as a longer precursor, DrrS+, which is rapidly processed to the mature and highly stable DrrS. We characterise, for the first time in mycobacteria, an RNA structural determinant involved in this extraordinary stability and we show how the addition of a few nucleotides can lead to acute destabilisation. Finally, we show how this RNA element can enhance expression of a heterologous gene. Thus, the element, as well as its destabilising derivatives may be employed to post-transcriptionally regulate gene expression in mycobacteria in combination with different promoter variants. Moreover, our findings will facilitate further investigations into the severely understudied topic of mycobacterial RNA biology and into the role that regulatory RNA plays in M. tuberculosis pathogenesis.

Small RNA Mcr11 requires the transcription factor AbmR for stable expression and regulates genes involved in the central metabolism of Mycobacterium tuberculosis

“…Protein coding mRNAs often can tolerate variable amounts of 5′ and 3′ flanking sequence because the signals for protein expression are provided immediately upstream and within the open reading frame (ORF). In contrast, expression of functional sRNAs may be more dependent on RNA chaperones and/or processing factors, as well as cis‐acting sequence elements at their transcriptional boundaries that are difficult to define at (Chae et al, ; Moores et al, ; Morita et al, ; Kriner, Sevostyanova, & Groisman, ; Shiflett et al, ). Although RNA chaperones have yet to be identified in mycobacteria, the roles of extended native 3′ sequences for expression of mycobacterial sRNAs warrant further investigation to determine whether mcr11 is exceptional or representative of a larger group of sRNAs with regards to expression and stability requirements (Ishikawa, Otaka, Maki, Morita, & Aiba, ; Johnson et al, ; Moores et al, ; Regnier & Hajnsdorf, ; Sauer & Weichenrieder, ; Shiflett et al, ; Olejniczak & Storz, ).…”

“…The discrete 3′ end of mature Mcr11 observed by Northern blot is consistent with a processed or precisely terminated RNA, as transcription through TSEs would otherwise result in significantly longer RNA products. Processing of the 3′ ends of mycobacterial sRNAs has been proposed (Tsai et al, ), and a recent report identified a hypoxia‐regulated mycobacterial sRNA that is extensively processed at its 3′ end (Moores et al, ). The observed size variants of specific sRNA species in response to host‐associated stress conditions (Arnvig & Young, ) provide further evidence for processing of sRNAs in mycobacteria.…”

“…However, the additional requirements for TB complex specific factors for stable Mcr11 expression were unexpected, and may have broader implications for understanding sRNA expression in Mtb. (Chae et al, 2011;Moores et al, 2017;Morita et al, 2015;Kriner, Sevostyanova, & Groisman, 2016;Shiflett et al, 2003).…”

“…Gene expression studies have provided critical insights into the regulation and function of proteins like transcription factors that modulate gene expression as Mtb adapts to the host environment during infection (Mvubu, Pillay, Gamieldien, Bishai, & Pillay, ; Schnappinger et al, ). The additional role of sRNAs in gene regulation is recognized in other bacteria (Waters & Storz, ), and several sRNAs whose expression is responsive to stress and/ or growth phase have been identified in mycobacteria (Arnvig et al, ; Arnvig & Young, ; DiChiara et al, ; Gerrick et al, ; Miotto et al, ; Moores, Riesco, Schwenk, & Arnvig, ; Namouchi et al, ; Pelly, Bishai, & Lamichhane, ; Solans et al, ; Tsai et al, ). It also has been observed that overexpression of some sRNAs leads to slow or delayed growth in mycobacteria (Arnvig & Young, ; Ignatov et al, ).…”

“…Hostassociated environmental cues that result in metabolic remodeling and a shift away from active growth toward a state of persistence include Schnappinger et al, 2003). The additional role of sRNAs in gene regulation is recognized in other bacteria (Waters & Storz, 2009), and several sRNAs whose expression is responsive to stress and/ or growth phase have been identified in mycobacteria (Arnvig et al, 2011;Arnvig & Young, 2009;DiChiara et al, 2010;Gerrick et al, 2018;Miotto et al, 2012;Moores, Riesco, Schwenk, & Arnvig, 2017;Namouchi et al, 2016;Pelly, Bishai, & Lamichhane, 2012;Solans et al, 2014;Tsai et al, 2013). It also has been observed that overexpression of some sRNAs leads to slow or delayed growth in mycobacteria (Arnvig & Young, 2009;Ignatov et al, 2015).…”