Identification of small RNAs in diverse bacterial species

Small, non-coding bacterial RNAs (sRNAs) have been shown to regulate a plethora of biological processes. Up until recently, most sRNAs had been identified and characterized in E. coli. However, in the past few years, dozens of sRNAs have been discovered in a wide variety of bacterial species. Whereas numerous sRNAs have been isolated or detected through experimental approaches, most have been identified in predictive bioinformatic searches. Recently developed computational tools have greatly facilitated the efficient prediction of sRNAs in diverse species. Although the number of known sRNAs has dramatically increased in recent years, many challenges in the identification and characterization of sRNAs lie ahead.     

Essential requirements for robust signaling in Hfq dependent small RNA networks

Bacteria possess networks of small RNAs (sRNAs) that are important for modulating gene expression. At the center of many of these sRNA networks is the Hfq protein. Hfq's role is to quickly match cognate sRNAs and target mRNAs from among a large number of possible combinations and anneal them to form duplexes. Here we show using a kinetic model that Hfq can efficiently and robustly achieve this difficult task by minimizing the sequestration of sRNAs and target mRNAs in Hfq complexes. This sequestration can be reduced by two non-mutually exclusive kinetic mechanisms. The first mechanism involves heterotropic cooperativity (where sRNA and target mRNA binding to Hfq is influenced by other RNAs bound to Hfq); this cooperativity can selectively decrease singly-bound Hfq complexes and ternary complexes with non-cognate sRNA-target mRNA pairs while increasing cognate ternary complexes. The second mechanism relies on frequent RNA dissociation enabling the rapid cycling of sRNAs and target mRNAs among different Hfq complexes; this increases the probability the cognate ternary complex forms before the sRNAs and target mRNAs degrade. We further demonstrate that the performance of sRNAs in isolation is not predictive of their performance within a network. These findings highlight the importance of experimentally characterizing duplex formation in physiologically relevant contexts with multiple RNAs competing for Hfq. The model will provide a valuable framework for guiding and interpreting these experiments.     

Regulatory RNAs in Haloferax volcanii

In organisms of all three domains of life, a plethora of sRNAs (small regulatory RNAs) exists in addition to the well-known RNAs such as rRNAs, tRNAs and mRNAs. Although sRNAs have been well studied in eukaryotes and in bacteria, the sRNA population in archaea has just recently been identified and only in a few archaeal species. In the present paper, we summarize our current knowledge about sRNAs and their function in the halophilic archaeon Haloferax volcanii. Using two different experimental approaches, 111 intergenic and 38 antisense sRNAs were identified, as well as 42 tRFs (tRNA-derived fragments). Observation of differential expression under various conditions suggests that these sRNAs might be active as regulators in gene expression like their bacterial and eukaryotic counterparts. The severe phenotypes observed upon deletion and overexpression of sRNA genes revealed that sRNAs are involved in, and important for, a variety of biological functions in H. volcanii and possibly other archaea. Investigation of the Haloferax Lsm protein suggests that this protein is involved in the archaeal sRNA pathway.     

Identification of bacterial small non-coding RNAs: experimental approaches

Almost 140 bacterial small RNAs (sRNAs; sometimes referred to as non-coding RNAs) have been discovered in the past six years. The majority of these sRNAs were discovered in Escherichia coli, and a smaller subset was characterized in other bacteria, many of which were pathogenic. Many of these genes were identified as a result of systematic screens using computational prediction of sRNAs and experimental-based approaches, including microarray and shotgun cloning. A smaller number of sRNAs were discovered by direct labeling or by functional genetic screens. Many of the discovered genes, ranging in size from 50 to 500 nucleotides, are conserved and located in intergenic regions, in-between open reading frames. The expression of many of these genes is growth phase dependent or stress related. As each search employed specific parameters, this led to the identification of genes with distinct characteristics. Consequently, unique sRNAs such as those that are species-specific, sRNA genes that are transcribed under unique conditions or genes located on the antisense strand of protein-encoding genes, were probably missed.     

Endogenous Small RNA Clusters in Plants

In plants, small RNAs（sRNAs） usually refer to non-coding RNAs（ncRNAs） with lengths of 20–24 nucleotides. sRNAs are involved in the regulation of many essential processes related to plant development and environmental responses. sRNAs in plants are mainly grouped into microRNAs（miRNAs） and small interfering RNAs（siRNAs）, and the latter can be further classified into trans-acting siRNAs（ta-siRNAs）, repeat-associated siRNAs（ra-siRNAs）, natural anti-sense siRNAs（nat-siRNAs）, etc. Many sRNAs exhibit a clustered distribution pattern in the genome. Here, we summarize the features and functions of cluster-distributed sRNAs, aimed to not only provide a thorough picture of sRNA clusters（SRCs） in plants, but also shed light on the identification of new classes of functional sRNAs.     

Acting antisense: plasmid- and chromosome-encoded sRNAs from Gram-positive bacteria

sRNAs that act by base pairing were first discovered in plasmids, phages and transposons, where they control replication, maintenance and transposition. Since 2001, however, computational searches were applied that led to the discovery of a plethora of sRNAs in bacterial chromosomes. Whereas the majority of these chromsome-encoded sRNAs have been investigated in Escherichia coli, Salmonella and other Gram-negative bacteria, only a few well-studied examples are known from Gram-positive bacteria. Here, the author summarizes our current knowledge on plasmid- and chromosome-encoded sRNAs from Gram-positive species, thereby focusing on regulatory mechanisms used by these RNAs and their biological role in complex networks. Furthermore, regulatory factors that control the expression of these RNAs will be discussed and differences between sRNAs from Gram-positive and Gram-negative bacteria highlighted. The main emphasis of this review is on sRNAs that act by base pairing (i.e., by an antisense mechanism). Thereby, both plasmid-encoded and chromosome-encoded sRNAs will be considered.     

The etiological agent of Lyme disease, Borrelia burgdorferi, appears to contain only a few small RNA molecules

Small regulatory RNAs (sRNAs) have recently been shown to be the main controllers of several regulatory pathways. The function of sRNAs depends in many cases on the RNA-binding protein Hfq, especially for sRNAs with an antisense function. In this study, the genome of Borrelia burgdorferi was subjected to different searches for sRNAs, including direct homology and comparative genomics searches and ortholog- and annotation-based search strategies. Two new sRNAs were found, one of which showed complementarity to the rpoS region, which it possibly controls by an antisense mechanism. The role of the other sRNA is unknown, although observed complementarities against particular mRNA sequences suggest an antisense mechanism. We suggest that the low level of sRNAs observed in B. burgdorferi is at least partly due to the presumed lack of both functional Hfq protein and RNase E activity.     

Identifying expression of new small RNAs by microarrays

Although a large number of small RNAs (sRNAs) have been discovered, it is very likely that the screens conducted so far have not reached saturation. Recently, many methods for predicting and identifying new sRNAs have been developed. However, it remains unclear what the total number of sRNAs within a genome is and how many types of sRNAs exist in plants and animals. In this article, combined methods of dynamic programming prediction, enrichment of sRNAs, and microarray analysis are developed to screen and evaluate a new class of sRNAs from introns of human, protein-encoding genes. The methods used by our laboratories to design capture probes and label enriched small RNAs are thoroughly described here. The microarray results show that our modified technologies are useful to enhance sensitivity and specificity of arrays, identify expression patterns within different cells, and discover differential expression of sRNAs during the differentiation process of bone marrow stem cells. Accordingly, the combination of computational prediction and microarray analysis may be a feasible and practical approach for profiling studies of both known and predicted small RNAs.     

Herbal decoctosome is a novel form of medicine

Traditionally, herbal medicine is consumed by drinking decoctions produced by boiling herbs with water. The functional components of the decoction are heat stable. Small RNAs(sRNAs) were reported as a new class of functional components in decoctions. However, the mechanisms by which sRNAs survive heat treatment of the decoction and enter cells are unclear.Previous studies showed that plant-derived exosome-like nanoparticles(ELNs), which we call botanosomes, could deliver therapeutic reagents in vivo. Here, we report that heat-stable decoctosomes(ELNs) from decoctions have more therapeutic effects than the decoctions in vitro and demonstrate therapeutic efficacy in vivo. Furthermore, sRNAs, such as HJT-sRNA-m7 and PGY-sRNA-6, in the decoctosome exhibit potent anti-fibrosis and anti-inflammatory effects, respectively. Decoctosome is comprised of lipids, chemical compounds, proteins, and s RNAs. A medical decoctosome mimic is called bencaosome. A single lipid sphinganine(d22:0) identified in the decoctosome was mixed and heated with the synthesized sRNAs to form the simplest bencaosome. This simple bencaosome structure was identified by critical micelle concentration(cmc) assay that sRNAs coassembled with sphinganine(d22:0) to form the lipid layers of vesicles. The heating process facilitates co-assembly of sRNAs and sphinganine(d22:0) until a steady state is reached. The artificially produced sphinganine-HJT-sRNA-m7 and sphinganinePGY-sRNA-6 bencaosomes could ameliorate bleomycin-induced lung fibrosis and poly(I:C)-induced lung inflammation, respectively, following oral administration in mice. Our study not only demonstrates that the herbal decoctosome may represent a combinatory remedy in precision medicine but also provides an effective oral delivery route for nucleic acid therapy.     

Dual-function RNA regulators in bacteria

The importance of small RNA (sRNA) regulators has been recognized across all domains of life. In bacteria, sRNAs typically control the expression of virulence and stress response genes via antisense base pairing with mRNA targets. Originally dubbed “non-coding RNAs,” a number of bacterial antisense sRNAs have been found to encode functional proteins. Although very few of these dual-function sRNAs have been characterized, they have been found in both gram-negative and gram-positive organisms. Among the few known examples, the functions and mechanisms of regulation by dual-function sRNAs are variable. Some dual-function sRNAs depend on the RNA chaperone Hfq for base pairing-dependent regulation (riboregulation); this feature appears so far exclusive to gram-negative bacterial sRNAs. Other variations can be found in the spatial organization of the coding region with respect to the riboregulation determinants. How the functions of encoded proteins relate to riboregulation is for the most part not understood. However, in one case it appears that there is physiological redundancy between protein and riboregulation functions. This mini-review focuses on the two best-studied bacterial dual-function sRNAs: RNAIII from Staphylococcus aureus and SgrS from Escherichia coli and includes a discussion of what is known about the structure, function and physiological roles of these sRNAs as well as what questions remain outstanding.