Identification of microRNA-size sRNAs Related to Salt Tolerance in <Emphasis Type="Italic">Spirulina platensis</Emphasis>

microRNAs (miRNAs) are endogenous non-coding small RNAs (sRNA) that play important regulatory functions in growth, development, and environmental stress response of eukaryotes. Currently, miRNA-size sRNA (msRNAs) was discovered in several prokaryotes through deep sequencing. However, no data are available on whether msRNAs exist in cyanobacteria and whether they regulate biological tolerance to salt stress. In this study, three small RNA libraries were constructed from control (0.02 M NaCl), medium- (0.3 M NaCl), and high-salt treatments (0.5 M NaCl) of Spirulina platensis. After sequencing using a high-throughput Illumina Solexa system, nine msRNAs with msRNA* were identified, and 21 candidate msRNAs showed significantly differential expression under salt-stress conditions. Seven of the selected msRNAs presented the consistent expression trends when compared with their deep sequencing results as verified by quantitative real-time polymerase chain reaction (qRT-PCR), demonstrating that the expression analyses for msRNAs according to small RNA sequencing data were reliable. Through computational identification, 33 target genes were predicted for 12 msRNAs in S. platensis; Gene Ontology (GO) and KEGG enrichment analyses revealed that the putative target genes were grouped into the categories of proteolysis, protein homotrimerization, glycosylation, ubiquinone biosynthetic process, DNA restriction-modification system, and polysaccharide catabolic process. Using proteomic analysis, two target proteins (glyceraldehyde-3-phosphate dehydrogenase and forkhead-associated (FHA) domain-containing protein) were differentially expressed under salt-stress conditions, which might be regulated by msRNAs. Our study demonstrates that msRNAs exist in S. platensis, and these msRNAs may have an important role in salt-stress responses; however, their functional significance requires further investigation.     

Regulation of small RNA stability: methylation and beyond

As central components of RNA silencing, small RNAs play diverse and important roles in many biological processes in eukaryotes. Aberrant reduction or elevation in the levels of small RNAs is associated with many developmental and physiological defects. The in vivo levels of small RNAs are precisely regulated through modulating the rates of their biogenesis and turnover. 2′-O-methylation on the 3′ terminal ribose is a major mechanism that increases the stability of small RNAs. The small RNA methyltransferase HUA ENHANCER1 (HEN1) and its homologs methylate microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs (piRNAs) in animals, and siRNAs in Drosophila. 3′ nucleotide addition, especially uridylation, and 3′-5′ exonucleolytic degradation are major mechanisms that turnover small RNAs. Other mechanisms impacting small RNA stability include complementary RNAs, cis-elements in small RNA sequences and RNA-binding proteins. Investigations are ongoing to further understand how small RNA stability impacts their accumulation in vivo in order to improve the utilization of RNA silencing in biotechnology and therapeutic applications.     

A particular set of small non-coding RNAs is bound to the distinctive Argonaute protein of Trypanosoma cruzi: Insights from RNA-interference deficient organisms

The study of small RNAs and Argonaute proteins in eukaryotes that are deficient in functional RNA interference could provide insights into novel functions of small RNAs. In this study we describe small non-coding RNAs bound to a distinctive Argonaute protein of Trypanosoma cruzi, TcPIWI-tryp. Co-immunoprecipitation of TcPIWI-tryp followed by deep sequencing of isolated RNA identified abundant small RNAs derived from rRNAs and tRNAs. The small RNA repertoire differed from that of the canonical Argonaute in organisms with functional RNA interference, which could indicate novel biological functions for TcPIWI-tryp in T. cruzi and other members of the trypanosomatid clade.     

The rapidly emerging ESBL-producing Escherichia coli O25-ST131 clone carries LPS core synthesis genes of the K-12 type

MicroRNAs (miRNAs) are important modulators of gene expression in eukaryotic cells. However RNAs of the same size in bacteria have not been specifically discussed previously. Here, we provide a library of miRNA-size RNAs (msRNAs), which were registered by deep sequencing in Streptococcus mutans. Bioinformatic analysis of the whole set revealed more than 900 individual msRNA species. The cellular content of selected msRNAs was verified by quantitative RT-PCR and Northern blotting. The high abundance and discrete size of the subset of registered msRNAs suggest their functional significance, although the precise biological role of the RNA species revealed in S.?mutans, which is one of the principle causative agents of dental caries, has to be elucidated.     

Small RNAs derived from snoRNAs

Small nucleolar RNAs (snoRNAs) guide RNA modification and are localized in nucleoli and Cajal bodies in eukaryotic cells. Components of the RNA silencing pathway associate with these structures, and two recent reports have revealed that a human and a protozoan snoRNA can be processed into miRNA-like RNAs. Here we show that small RNAs with evolutionary conservation of size and position are derived from the vast majority of snoRNA loci in animals (human, mouse, chicken, fruit fly), Arabidopsis, and fission yeast. In animals, sno-derived RNAs (sdRNAs) from H/ACA snoRNAs are predominantly 20–24 nucleotides (nt) in length and originate from the 3′ end. Those derived from C/D snoRNAs show a bimodal size distribution at ∼17–19 nt and >27 nt and predominantly originate from the 5′ end. SdRNAs are associated with AGO7 in Arabidopsis and Ago1 in fission yeast with characteristic 5′ nucleotide biases and show altered expression patterns in fly loquacious and Dicer-2 and mouse Dicer1 and Dgcr8 mutants. These findings indicate that there is interplay between the RNA silencing and snoRNA-mediated RNA processing systems, and that sdRNAs comprise a novel and ancient class of small RNAs in eukaryotes.     

The Fascinating World of RNA Interference

Micro- and short-interfering RNAs represent small RNA family that are recognized as critical regulatory species across the eukaryotes. Recent high-throughput sequencing have revealed two more hidden players of the cellular small RNA pool. Reported in mammals and Caenorhabditis elegans respectively, these new small RNAs are named piwi-interacting RNAs (piRNAs) and 21U-RNAs. Moreover, small RNAs including miRNAs have been identified in unicellular alga Chlamydomonas reinhardtii, redefining the earlier concept of multi-cellularity restricted presence of these molecules. The discovery of these species of small RNAs has allowed us to understand better the usage of genome and the number of genes present but also have complicated the situation in terms of biochemical attributes and functional genesis of these molecules. Nonetheless, these new pools of knowledge have opened up avenues for unraveling the finer details of the small RNA mediated pathways.     

SINE RNA induces severe developmental defects in Arabidopsis thaliana and interacts with HYL1 (DRB1), a key member of the DCL1 complex

The proper temporal and spatial expression of genes during plant development is governed, in part, by the regulatory activities of various types of small RNAs produced by the different RNAi pathways. Here we report that transgenic Arabidopsis plants constitutively expressing the rapeseed SB1 SINE retroposon exhibit developmental defects resembling those observed in some RNAi mutants. We show that SB1 RNA interacts with HYL1 (DRB1), a double-stranded RNA-binding protein (dsRBP) that associates with the Dicer homologue DCL1 to produce microRNAs. RNase V1 protection assays mapped the binding site of HYL1 to a SB1 region that mimics the hairpin structure of microRNA precursors. We also show that HYL1, upon binding to RNA substrates, induces conformational changes that force single-stranded RNA regions to adopt a structured helix-like conformation. Xenopus laevis ADAR1, but not Arabidopsis DRB4, binds SB1 RNA in the same region as HYL1, suggesting that SINE RNAs bind only a subset of dsRBPs. Consistently, DCL4-DRB4-dependent miRNA accumulation was unchanged in SB1 transgenic Arabidopsis, whereas DCL1-HYL1-dependent miRNA and DCL1-HYL1-DCL4-DRB4-dependent tasiRNA accumulation was decreased. We propose that SINE RNA can modulate the activity of the RNAi pathways in plants and possibly in other eukaryotes.     

Roles and Programming of Arabidopsis ARGONAUTE Proteins during Turnip Mosaic Virus Infection

In eukaryotes, ARGONAUTE proteins (AGOs) associate with microRNAs (miRNAs), short interfering RNAs (siRNAs), and other classes of small RNAs to regulate target RNA or target loci. Viral infection in plants induces a potent and highly specific antiviral RNA silencing response characterized by the formation of virus-derived siRNAs. Arabidopsis thaliana has ten AGO genes of which AGO1, AGO2, and AGO7 have been shown to play roles in antiviral defense. A genetic analysis was used to identify and characterize the roles of AGO proteins in antiviral defense against Turnip mosaic virus (TuMV) in Arabidopsis. AGO1, AGO2 and AGO10 promoted anti-TuMV defense in a modular way in various organs, with AGO2 providing a prominent antiviral role in leaves. AGO5, AGO7 and AGO10 had minor effects in leaves. AGO1 and AGO10 had overlapping antiviral functions in inflorescence tissues after systemic movement of the virus, although the roles of AGO1 and AGO10 accounted for only a minor amount of the overall antiviral activity. By combining AGO protein immunoprecipitation with high-throughput sequencing of associated small RNAs, AGO2, AGO10, and to a lesser extent AGO1 were shown to associate with siRNAs derived from silencing suppressor (HC-Pro)-deficient TuMV-AS9, but not with siRNAs derived from wild-type TuMV. Co-immunoprecipitation and small RNA sequencing revealed that viral siRNAs broadly associated with wild-type HC-Pro during TuMV infection. These results support the hypothesis that suppression of antiviral silencing during TuMV infection, at least in part, occurs through sequestration of virus-derived siRNAs away from antiviral AGO proteins by HC-Pro. These findings indicate that distinct AGO proteins function as antiviral modules, and provide a molecular explanation for the silencing suppressor activity of HC-Pro.     

Higher-order structure in the 3′-terminal domain VI of the 23 S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus

An experimental approach was used to determine, and compare, the higher-order structure within domain VI of the 23 S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus. This domain, which encompasses approximately 300 nucleotides at the 3′ end of the RNAs, consists of two large subdomains. The 5′ subdomain has been conserved during evolution and appears to be functionally important for the binding of the EF-1 · GTP · aminoacyl-tRNA complex in eukaryotes. The 3′ subdomain has diverged widely between eubacteria and eukaryotes and has produced the 4.5 S RNA in the chloroplast ribosomes of flowering plants.The structure of domain VI within the eubacterial RNAs was probed with chemical reagents in order to establish the degree of stacking and/or accessibility of each adenosine, cytidine and guanosine residue; the double-helical segments were localized with the cobra venom ribonuclease from Naja naja oxiana, and the relatively unstructured and accessible sequences were detected with the single-strand-specific ribonucleases A, T₁ and T₂. The data enabled the three secondary structural models, proposed for the E. coli 23 S RNAs, to be examined critically and it was concluded that many of their structural features are correct. Various differences between the models were considered and evidence is provided for additional structuring in the RNA including the stacking of juxtaposed purines into double helices. The 5′ subdomain constitutes a compact and resistant structure whereas the 3′ subdomain is relatively accessible and contains most of the potential protein binding sites. Moreover, comparison of our results with the published results on 4.5 S RNA suggests that the latter forms essentially the same structure as the 3′ subdomain, in contrast to earlier conclusions.A high level of structural conservation has occurred throughout the RNA domain during the evolution of the Gram negative and Gram positive bacteria although the thermophile was generally more stable at base-pairs adjacent to the terminal loops.     

Characterization and application of small RNAs and RNA silencing mechanisms in fungi

Although extensively cataloged and functionally diverse in plants and animals, the role and targets of small RNAs remain mostly uncharacterized in filamentous fungi. To date, much of the knowledge of small RNAs in filamentous fungi has been derived from studies of a limited group of fungi, most notably in Neurospora crassa. While most of the recently discovered classes of small RNAs appear to be unique to fungi some are commonly found in eukaryotes. It is noteworthy that the RNA silencing protein machinery involved in small RNA biogenesis has also diverged greatly, particularly within filamentous fungi, and may explain the diversity of small RNA classes. In this review, we summarize important classes of eukaryotic small RNAs and provide a current analysis of the RNA silencing machinery based on available fungal genome sequences. Finally, we discuss opportunities for exploiting knowledge of small RNAs and RNA silencing for practical application such as engineering plants resistant to fungal pathogens.     

Characterization and application of small RNAs and RNA silencing mechanisms in fungi

Although extensively cataloged and functionally diverse in plants and animals, the role and targets of small RNAs remain mostly uncharacterized in filamentous fungi. To date, much of the knowledge of small RNAs in filamentous fungi has been derived from studies of a limited group of fungi, most notably in Neurospora crassa. While most of the recently discovered classes of small RNAs appear to be unique to fungi some are commonly found in eukaryotes. It is noteworthy that the RNA silencing protein machinery involved in small RNA biogenesis has also diverged greatly, particularly within filamentous fungi, and may explain the diversity of small RNA classes. In this review, we summarize important classes of eukaryotic small RNAs and provide a current analysis of the RNA silencing machinery based on available fungal genome sequences. Finally, we discuss opportunities for exploiting knowledge of small RNAs and RNA silencing for practical application such as engineering plants resistant to fungal pathogens.     

Small RNA Profile in Moso Bamboo Root and Leaf Obtained by High Definition Adapters

Moso bamboo (Phyllostachy heterocycla cv. pubescens L.) is an economically important fast-growing tree. In order to gain better understanding of gene expression regulation in this important species we used next generation sequencing to profile small RNAs in leaf and roots of young seedlings. Since standard kits to produce cDNA of small RNAs are biased for certain small RNAs, we used High Definition adapters that reduce ligation bias. We identified and experimentally validated five new microRNAs and a few other small non-coding RNAs that were not microRNAs. The biological implication of microRNA expression levels and targets of microRNAs are discussed.