Identification of small RNAs in Mycobacterium smegmatis using heterologous Hfq

Abstract

Gene regulation by small RNAs (sRNAs) has been extensively studied in various bacteria. However, the presence and roles of sRNAs in mycobacteria remain largely unclear. Immunoprecipitation of RNA chaperone Hfq to enrich for sRNAs is one of the effective methods to isolate sRNAs. However, the lack of an identified mycobacterial hfq restricts the feasibility of this approach. We developed a novel method that takes advantage of the conserved inherent sRNAs-binding capability of heterologous Hfq from Escherichia coli to enrich sRNAs from Mycobacterium smegmatis, a model organism for studying Mycobacterium tuberculosis. We validated 12 trans-encoded and 12 cis-encoded novel sRNAs in M. smegmatis. Many of these sRNAs are differentially expressed at exponential phase compared with stationary phase, suggesting that sRNAs are involved in the growth of mycobacteria. Intriguingly, five of the cis-encoded novel sRNAs target known transposases. Phylogenetic conservation analysis shows that these sRNAs are pathogenicity dependent. We believe that our findings will serve as an important reference for future analysis of sRNAs regulation in mycobacteria and will contribute significantly to the development of sRNAs prediction programs. Moreover, this novel method of using heterologous Hfq for sRNAs enrichment can be of general use for the discovery of bacterial sRNAs in which no endogenous Hfq is identified.

Biochemical & Biophysical Studies of an Hfq Homolog from a Deep-branching Bacterium

“…The IC50 and K i for these nanoRNAs are reported in Table 2.1. The pattern of variance in the binding affinities suggests that Hfq preferentially interacts with uracil, as is known from many previous in vitro studies [40,53,54,55,56]. More specifically, the data indicate that a uracil base at the 3' end of the nanoRNA is important for high affinity interactions with Hfq, while a cytosine in the fifth position of the nanoRNA significantly decreases the affinity between Tma Hfq and the nanoRNA.…”

“…The L4 loop is located on the "distal" face of Sm oligomers and contributes to differences in the surface electrostatic potential. For instance, the electrostatic potential of the distal face of an archaeal Hfq from Methanocaldoccocus jannaschii is predominantly negative [54], but is positive for the Hfq from E. coli [50]. Differences in electrostatic potential between various Sm proteins are illustrated in Figure 1.5.…”

“…Hfq can be co-immunoprecipitated with Hfq specific antibodies [51,52] (such as the rabbit antiTma Hfq pAb described in Appendix 2) or with antibodies specific to a protein-tag genetically added to a recombinantly expressed Hfq [3,53,54,55]. These co-immunoprecipitated Hfqbinding RNAs can then be identified using well-established protocols, such as deep sequencing (also known as NGS) [3,54,55] or microarrays [51,52].…”

“…These co-immunoprecipitated Hfqbinding RNAs can then be identified using well-established protocols, such as deep sequencing (also known as NGS) [3,54,55] or microarrays [51,52].…”

Mycobacterium tuberculosis Metabolism

“…Paramount among these are the signals and signaling mechanisms that regulate metabolic adaptations to different host environments, as well as the transcriptional and posttranslational events that enable the mycobacterial metabolic network to tolerate the perturbations that must inevitably be encountered during host infection. There is increasing evidence that noncoding RNAs are key to the regulation and function of certain metabolic pathways in M. tuberculosis (Arnvig et al 2011;Golby et al 2013;Li et al 2013). Moreover, recent observations that specific intergenic regions are essential for mycobacterial survival (Zhang et al 2012) or are involved in the emergence of drug resistance (Zhang et al 2013a) hint at a further level of regulation that remains largely unexplored.…”