Membrane RNAs in bacteria

OLE (Ornate Large Extremophile) RNAs constitute a family of large, well-conserved noncoding transcripts of unknown function. In this issue of Molecular Microbiology, Kirsten Block, Ron Breaker and co-workers begin to decipher some of the functional mysteries surrounding OLE RNA. This class of RNA is approximately 610 nucleotides in length, with an average nucleotide identity of 65%. The OLE RNA family was discovered using comparative sequence analysis of bacterial genomes. It was found in Bacillus halodurans and in extremophiles of the Clostridiae order. So far, OLE RNA is confined to anaerobic bacteria from the environment and the human gut. The large number of well-conserved OLE RNA sequences has made it possible to use sequence co-variation analysis to accurately predict OLE RNA secondary structures (Puerta-Fernandez et al., 2006; Tseng et al., 2009; Weinberg et al., 2007; Weinberg et al., 2009). OLE RNAs are among the most complex noncoding RNAs currently known to exist in bacteria (Weinberg et al., 2009). OLE RNA is part of a large and complex transcriptional unit (Figure 1A) that contains 10 other genes including OAP (OLE-Associated Protein). The OAP protein is encoded by a gene located just downstream of the OLE RNA gene. To begin to understand the biological role of OLE RNA, Block and co-workers analyzed the interaction between the RNA and OAP of B. halodurans in vitro. Using gel shift assays, they found that OAP and OLE form a Ribo-Nucleo-protein Particle (RNP). By systematically removing domains of the OLE RNA, they were able to map the binding site of OAP in the RNA. This analysis yielded a sequence motif that is highly conserved. The fact that OLE RNA and OAP are co-transcribed and form an RNP suggests that this interaction is functionally important. Figure 1 A) Schematic representation of the transcript containing OLE RNA (red) as well as the OLE-associate protein (OAP, green) as transcribed from genomic DNA. OLE is co-transcribed with OAP. B) Rank ordered plot of the RNA expression levels (as measured by ... OAP has several transmembrane domains, raising the possibility that OAP is a membrane protein that recruits OLE RNA to the cell membrane. To test this, the authors performed Fluorescent In Situ Hybridization (FISH) on OLE RNA to determine its sub-cellular localization. Indeed, they found that OLE RNA localized to the membrane in E. coli. The localization of OLE to the membrane raises the possibility that it has a novel cellular function. The remarkable “ornate” nature of OLE RNA’s secondary structure is so highly conserved that it could be deduced solely by co-variation analysis. The conserved structure indicates that the RNA has functional significance. Further evidence for this conjecture comes from the analysis of global expression levels of B. halodurans RNAs. Breaker and co-workers measured the relative expression level of all B. halodurans transcripts using deep sequencing and found that OLE RNA is the 11th most abundant RNA in the cell. Its expression level is 10% of that of the ubiquitous Signal Recognition Particle (SRP). We plot in Figure 1B data provided in the supplement of the manuscript, which reports on the relative expression of all transcripts in B. halodurans. The fact that OLE RNA is one of the most highly expressed RNAs provides further support for a central functional role. Bacteria expressing OLE must expend significant energy producing such large quantities of RNA, meaning that it must confer an evolutionary advantage. Furthermore, because OLE is found in extremophiles, it is possible that its function is related to the adaptation to extreme environments. Combined with its recruitment to the membrane by OAP, it is likely that OLE may have a novel function in the cell. B. halodurans has approximately 4200 genes and as is illustrated in Figure 1B, 4000 of those genes are identified by deep sequencing RNA transcripts. The result reveals the abundance of RNAs that are continuously produced. The fact that some of the most abundant RNAs are non-coding (e.g. OLE, SRP, tmRNA and RNASE P) provides a clear illustration of the functional importance of non-coding RNA in the cell. This study also reveals how inexpensive next generation sequencing is revolutionizing the field of molecular microbiology. Indeed, OLE RNA was identified from an analysis of thousands of bacterial genomes. Furthermore, it is now possible to characterize an entire bacterial transcriptome in a single experiment (Figure 1B). We can therefore expect to discover many new non-coding and functionally important RNAs with these new techniques. The challenge will be to unravel their biological function.