Navigation through the twists and turns of RNA sequencing technologies: Application to bacterial regulatory RNAs☆
Introduction
The continuous advances in the characterization of bacterial small regulatory RNAs (sRNAs) have allowed the appreciation of their diversity regarding their origins, sizes and functions [1,2]. Usually, the transcription of sRNAs relies on two-component systems and/or transcriptional factors responding directly to environmental stimuli or metabolites [2]. This heterogeneous group of RNAs includes not only bone fide sRNAs, acting in trans and having their own promoter and transcriptional terminator, but also RNA fragments released from precursor RNAs. Typically, these fragments are the result of an RNase-dependent cleavage (e.g. RNase E, RNase III) of 3′ untranslated region (UTR) of mRNAs [3], 3′ external transcribed spacer of tRNA transcripts [4] or sRNAs, which are processed to give mature forms. For example, ArcZ sRNA is transcribed as a 120 nt-long fragment in Escherichia coli and then processed into two shorter forms, a low abundant (88 nts) and a stable (55 nts) fragments, sharing the same 3′ end [5]. The genesis of an sRNA (proper transcription or maturation) can be easily discriminated by analyzing their distinctive 5′ ends. Primary RNAs, which are produced from their own promoter, have a 5′ triphosphorylated (5′PPP) end, while secondary RNAs which are processed from a precursor transcript, have a 5′ monophosphorylated (5′P) end. Hence, 5′ ends are characterized as transcription start sites (TSS) and processing start sites (PSS), respectively.
sRNAs do not only differ in their origins but also in their lengths. They are commonly described as ranging from 50 to 500 nts. However, recent findings revealed that they are even more heterogeneous in size, from dozens to over a thousand nucleotides. Indeed, very small RNA fragments (such as tRNA-derived fragments [6]) and large transcripts (e.g. 1116 nt-long RsaC sRNA in Staphylococcus aureus [7]) have been described as trans-acting regulators.
sRNAs act mainly by forming non-contiguous pairings with their target mRNAs, thus regulating their translation and/or stability. Chaperone proteins like Hfq or ProQ are often involved in sRNAs stability and/or functions [8,9]. Remarkably, an sRNA can regulate many mRNA targets, constituting its targetome, and one target can be regulated by numerous sRNAs. As a result, sRNAs post-transcriptionally control the expression of their multiple mRNA targets involved in all kinds of cellular processes and can even regulate their own transcription factors via feedback loops. They are often non-coding, meaning that they do not bear ribosome binding signals and in frame start and stop codons, the key features of coding sequences. Yet few are named bifunctional or dual-function sRNAs, as they encode peptides like SgrS in E. coli and RNAIII in S. aureus [10], coding for the SgrT peptide and the Hld toxin, respectively. In addition, a few sRNAs buffer other sRNA transcripts [4,11] or cellular proteins [12,13]. In a nutshell, sRNAs integrate various internal and/or external signals and are at the center of complex regulatory networks, impacting every aspect of bacterial physiology/virulence.
This review will provide an overview of cutting-edge technologies/approaches for the complete characterization of bacterial sRNAs, their whole targetome and their cellular functions. Due to the profusion of available algorithms/methods, we provide here a non-comprehensive list, and we sincerely apologize to authors of uncited works.
Section snippets
Identification of sRNA pool in a bacterial genome
Historically, sRNAs have been mainly identified by computational searches (potential promoters and rho-independent terminators in intergenic region), comparative genomics (sequence conservation) or co-purification with RNA-binding proteins such as Hfq and CsrA/RsmA [14,15]. However, the introduction of tiling microarrays and RNA sequencing (RNA-seq)-based approaches has revolutionized our understanding of bacterial transcriptomes, including sRNAs. With the development of next-generation
Characterization of sRNAs at a single molecule or a global scale
Full sRNAs characterization is a prerequisite for the experimental design to unveil their targetome and functions (Section 4). For example, a careful determination of the 5′ and 3′ end of sRNAs (and their mRNA targets) is required for cloning and mutagenesis purposes and, consequently, for the characterization of sRNA-dependent regulatory mechanisms. This is also important for the study of sRNAs genesis and expression.
In the following paragraphs, we will describe suitable methods allowing the
Characterization of their targetome
Several features make the characterization of sRNAs targetome difficult: (1) trans-acting sRNAs often interact with multiple target mRNAs (e.g. >30 targets for GcvB sRNA in E. coli [64]) using limited and non-contiguous sequence complementarities; (2) a single sRNA can bear multiple pairing sites, also named “seed” sequences; (3) RNA chaperone proteins such as Hfq [9] and ProQ [8] are often described as promoting the pairing between an sRNA and its partners especially in Enterobacteriaceae.
Conclusion
The recent and considerable evolution in high-throughput sequencing technologies has not only uncovered knowledge on sRNAs characterization and expression, but also on their cellular functions. This enables to build vast and complex regulatory networks integrating perceived stimuli and the impact on bacterial physiology. To go further, cell-to-cell diversity should be considered. Indeed, heterogeneous behavior between bacterial individuals is usually observed, notably in specific conditions
CRediT authorship contribution statement
Emma Desgranges: Writing - review & editing. Isabelle Caldelari: Writing - review & editing. Stefano Marzi: Writing - review & editing. David Lalaouna: Writing - review & editing, Methodology.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank Pascale Romby (PR) for helpful advice and discussions. We also thank the Integrative Molecular and Cellular Biology (IMCBio) graduate school.
Funding
This work was supported by the “Agence Nationale de la Recherche” (ANR, Grant ANR-16-CE11-0007-01, RIBOSTAPH, and ANR-18-CE12-0025-04, CoNoCo, to PR). It has also been published under the framework of the labEx NetRNA ANR-10-LABX-0036 and of ANR-17-EURE-0023 (to PR), as funding from the state managed by ANR as part of the investments for the future program. DL was supported by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No.
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This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.