Ms1 RNA Interacts With the RNA Polymerase Core in Streptomyces coelicolor and Was Identified in Majority of Actinobacteria Using a Linguistic Gene Synteny Search

Abstract

Bacteria employ small non-coding RNAs (sRNAs) to regulate gene expression. Ms1 is an sRNA that binds to the RNA polymerase (RNAP) core and affects the intracellular level of this essential enzyme. Ms1 is structurally related to 6S RNA that binds to a different form of RNAP, the holoenzyme bearing the primary sigma factor. 6S RNAs are widespread in the bacterial kingdom except for the industrially and medicinally important Actinobacteria. While Ms1 RNA was identified in Mycobacterium, it is not clear whether Ms1 RNA is present also in other Actinobacteria species. Here, using a computational search based on secondary structure similarities combined with a linguistic gene synteny approach, we identified Ms1 RNA in Streptomyces. In S. coelicolor, Ms1 RNA overlaps with the previously annotated scr3559 sRNA with an unknown function. We experimentally confirmed that Ms1 RNA/scr3559 associates with the RNAP core without the primary sigma factor HrdB in vivo. Subsequently, we applied the computational approach to other Actinobacteria and identified Ms1 RNA candidates in 824 Actinobacteria species, revealing Ms1 RNA as a widespread class of RNAP binding sRNAs, and demonstrating the ability of our multifactorial computational approach to identify weakly conserved sRNAs in evolutionarily distant genomes.

RIP-seq reveals RNAs that interact with RNA polymerase and primary sigma factors in bacteria

“…We confirmed that scr3559 sRNA is an Ms1 homolog in S. coelicolor ( 37 ) (Figure 6A and B ) and RIP-seq also verified the first nucleotide of scr3559 (Figure 6E and F ) which we had determined by 5′ RACE previously ( 37 ). In exponential phase, scr3559 strongly interacted with RNAP (∼310-fold enrichment) and did not interact with HrdB (∼2-fold enrichment, Figure 6A and C , Supplementary Table S5 ).…”

“…Nevertheless, mycobacteria are rod-shaped and unicellular, while streptomyces have a complex life cycle that starts with spore germination, then proceeds to a filamentous and highly branched vegetative (primary) mycelium, followed by formation of a secondary mycelium and eventually spores. Recently, there were contradictory results describing the presence of 6S RNA or an Ms1 homolog in Streptomyces coelicolor ( 30 , 31 , 37 ).…”

“…Therefore, we also focused on RNAs associated with the transcriptional machinery in S. coelicolor A3(2) hrdB- HA, a strain in which the primary σ factor (HrdB) is endogenously tagged with hemagglutinin (HA) ( 38 ). We had previously shown that the anti-HA antibody immunoprecipitated the primary σ factor holoenzyme (HrdB–HA in complex with RNAP) ( 37 ). Here, we performed RNAP and HrdB-HA RIP-seq from exponential and stationary phases of growth (42 h and 66 h after germination, respectively, Figure 6 ; Supplementary Table S5 , for details, see the Supplementary material ).…”

“…( E ) RIP-seq using S. coelicolor cells in stationary phase revealed the scr3559 transcript (Ms1 homolog) to strongly enrich with RNAP, but there are also some reads mapping to scr3559 RNA in HrdB RIP-seq samples (in green). ( F ) The first nucleotide of scr3559 RNA that associates with RNAP in stationary phase of growth corresponds to the first nucleotide of the scr3559 transcript as defined by 5′RACE using total RNA and published previously ( 37 ). ( G ) scr0792, a novel transcript from the intergenic region between the genes SCO0791 and SCO0792 binds to RNAP in stationary phase of growth in S. coelicolor .…”

“…Recently, putative Ms1 RNAs homologs have been found among many actinobacteria, for example in Streptomyces coelicolor ( 36 , 37 ). In some actinobacteria, such as corynebacteria, neither 6S RNA nor Ms1 have been discovered so far ( 37 ) raising the question whether any similar RNA exists in these species. It is also unknown if both Ms1 and 6S RNA are present in Streptomyces coelicolor .…”