Assessing small RNA profiles in potato diploid hybrid and its resynthesized allopolyploid reveals conserved abundance with distinct genomic distribution.

KEY MESSAGE: The shock produced by the allopolyploidization process on a potato interspecific diploid hybrid displays a non-random remobilization of the small RNAs profile on a variety of genomic features. Allopolyploidy, a complex process involving interspecific hybridization and whole genome duplication, significantly impacts plant evolution, leading to the emergence of novel phenotypes. Polyploids often present phenotypic nuances that enhance adaptability, enabling them to compete better and occasionally to colonize new habitats. Whole-genome duplication represents a genomic "shock" that can trigger genetic and epigenetic changes that yield novel expression patterns. In this work, we investigate the polyploidization effect on a diploid interspecific hybrid obtained through the cross between the cultivated potato Solanum tuberosum and the wild potato Solanum kurtzianum, by assessing the small RNAs (sRNAs) profile of the parental diploid hybrid and its derived allopolyploid. Small RNAs are key components of the epigenetic mechanisms involved in silencing by RNA-directed DNA Methylation (RdDM). A sRNA sequencing (sRNA-Seq) analysis was performed to individually profile the 21 to 22 nucleotide (21 to 22-nt) and 24-nt sRNA size classes due to their unique mechanism of biogenesis and mode of function. The composition and distribution of different genomic features and differentially accumulated (DA) sRNAs were evaluated throughout the potato genome. We selected a subset of genes associated with DA sRNAs for messenger RNA (mRNA) expression analysis to assess potential impacts on the transcriptome. Interestingly, we noted that 24-nt DA sRNAs that exclusively mapped to exons were correlated with differentially expressed mRNAs between genotypes, while this behavior was not observed when 24-nt DA sRNAs were mapped to intronic regions. These findings collectively emphasize the nonstochastic nature of sRNA remobilization in response to the genomic shock induced by allopolyploidization.

A comprehensive analysis of the Bencao (herbal) small RNA Atlas reveals novel RNA therapeutics for treating human diseases.

Cross-kingdom herbal miRNA was first reported in 2012. Using a modified herbal extraction protocol, we obtained 73,677,287 sequences by RNA-seq from 245 traditional Chinese Medicine (TCM), of which 20,758,257 were unique sequences. We constructed a Bencao (herbal) small RNA (sRNA) Atlas ( http://bencao.bmicc.cn ), annotated the sequences by sequence-based clustering, and created a nomenclature system for Bencao sRNAs. The profiles of 21,757 miRNAs in the Atlas were highly consistent with those of plant miRNAs in miRBase. Using software tools, our results demonstrated that all human genes might be regulated by sRNAs from the Bencao sRNA Atlas, part of the predicted human target genes were experimentally validated, suggesting that Bencao sRNAs might be one of the main bioactive components of herbal medicines. We established roadmaps for oligonucleotide drugs development and optimization of TCM prescriptions. Moreover, the decoctosome, a lipo-nano particle consisting of 0.5%-2.5% of the decoction, demonstrated potent medical effects. We propose a Bencao (herbal) Index, including small-molecule compounds (SM), protein peptides (P), nucleic acid (N), non-nucleic and non-proteinogenic large-molecule compounds (LM) and elements from Mendeleev's periodic table (E), to quantitatively measure the medical effects of botanic medicine. The Bencao sRNA Atlas is a resource for developing gene-targeting oligonucleotide drugs and optimizing botanical medicine, and may provide potential remedies for the theory and practice of one medicine.

Paternal environmental exposure-induced spermatozoal small noncoding RNA alteration meditates the intergenerational epigenetic inheritance of multiple diseases.

Studies of human and mammalian have revealed that environmental exposure can affect paternal health conditions as well as those of the offspring. However, studies that explore the mechanisms that meditate this transmission are rare. Recently, small noncoding RNAs (sncRNAs) in sperm have seemed crucial to this transmission due to their alteration in sperm in response to environmental exposure, and the methodology of microinjection of isolated total RNA or sncRNAs or synthetically identified sncRNAs gradually lifted the veil of sncRNA regulation during intergenerational inheritance along the male line. Hence, by reviewing relevant literature, this study intends to answer the following research concepts: (1) paternal environmental factors that can be passed on to offspring and are attributed to spermatozoal sncRNAs, (2) potential role of paternal spermatozoal sncRNAs during the intergenerational inheritance process, and (3) the potential mechanism by which spermatozoal sncRNAs meditate intergenerational inheritance. In summary, increased attention highlights the hidden wonder of spermatozoal sncRNAs during intergenerational inheritance. Therefore, in the future, more studies should focus on the origin of RNA alteration, the target of RNA regulation, and how sncRNA regulation during embryonic development can be sustained even in adult offspring.

Paternal environmental exposure-induced spermatozoal small noncoding RNA alteration meditates the intergenerational epigenetic inheritance of multiple diseases / 医学前沿

Studies of human and mammalian have revealed that environmental exposure can affect paternal health conditions as well as those of the offspring. However, studies that explore the mechanisms that meditate this transmission are rare. Recently, small noncoding RNAs (sncRNAs) in sperm have seemed crucial to this transmission due to their alteration in sperm in response to environmental exposure, and the methodology of microinjection of isolated total RNA or sncRNAs or synthetically identified sncRNAs gradually lifted the veil of sncRNA regulation during intergenerational inheritance along the male line. Hence, by reviewing relevant literature, this study intends to answer the following research concepts: (1) paternal environmental factors that can be passed on to offspring and are attributed to spermatozoal sncRNAs, (2) potential role of paternal spermatozoal sncRNAs during the intergenerational inheritance process, and (3) the potential mechanism by which spermatozoal sncRNAs meditate intergenerational inheritance. In summary, increased attention highlights the hidden wonder of spermatozoal sncRNAs during intergenerational inheritance. Therefore, in the future, more studies should focus on the origin of RNA alteration, the target of RNA regulation, and how sncRNA regulation during embryonic development can be sustained even in adult offspring.

Methodologies for Discovery and Quantitative Profiling of sRNAs in Potato.

Small RNAs (sRNAs) are short noncoding RNAs involved in the regulation of a wide range of biological processes in plants. Advances in high-throughput sequencing and development of new computational tools had facilitated the discovery of different classes of sRNAs, their quantification, and elucidation of their functional role in gene expression regulation by target transcript predictions. The workflow presented here allows identification of different sRNA species: known and novel potato miRNAs, and their sequence variants (isomiRs), as well as identification of phased small interfering RNAs (phasiRNAs). Moreover, it includes steps for differential expression analysis to search for regulated sRNAs across different tested biological conditions. In addition, it describes two different methods for predicting sRNA targets, in silico prediction, and degradome sequencing data analysis. All steps of the workflow are written in a clear and user-friendly way; thus they can be followed also by the users with minimal bioinformatics knowledge. We also included several in-house scripts together with valuable notes to facilitate data (pre)processing steps and to reduce the analysis time.

Ubiquitin-dependent Argonauteprotein MEL1 degradation is essential for rice sporogenesis and phasiRNA target regulation.

MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice (Oryza sativa) Argonaute (AGO) protein, has been reported to function specifically at premeiotic and meiotic stages of germ cell development and is associated with a novel class of germ cell-specific small noncoding RNAs called phased small RNAs (phasiRNAs). MEL1 accumulation is temporally and spatially regulated and is eliminated after meiosis. However, the metabolism and turnover (i.e. the homeostasis) of MEL1 during germ cell development remains unknown. Here, we show that MEL1 is ubiquitinated and subsequently degraded via the proteasome pathway in vivo during late sporogenesis. Abnormal accumulation of MEL1 after meiosis leads to a semi-sterile phenotype. We identified a monocot-specific E3 ligase, XBOS36, a CULLIN RING-box protein, that is responsible for the degradation of MEL1. Ubiquitination at four K residues at the N terminus of MEL1 by XBOS36 induces its degradation. Importantly, inhibition of MEL1 degradation either by XBOS36 knockdown or by MEL1 overexpression prevents the formation of pollen at the microspore stage. Further mechanistic analysis showed that disrupting MEL1 homeostasis in germ cells leads to off-target cleavage of phasiRNA target genes. Our findings thus provide insight into the communication between a monocot-specific E3 ligase and an AGO protein during plant reproductive development.

Locus-specific paramutation in Zea mays is maintained by a PICKLE-like chromodomain helicase DNA-binding 3 protein controlling development and male gametophyte function.

Paramutations represent directed and meiotically-heritable changes in gene regulation leading to apparent violations of Mendelian inheritance. Although the mechanism and evolutionary importance of paramutation behaviors remain largely unknown, genetic screens in maize (Zea mays) identify five components affecting 24 nucleotide RNA biogenesis as required to maintain repression of a paramutant purple plant1 (pl1) allele. Currently, the RNA polymerase IV largest subunit represents the only component also specifying proper development. Here we identify a chromodomain helicase DNA-binding 3 (CHD3) protein orthologous to Arabidopsis (Arabidopsis thaliana) PICKLE as another component maintaining both pl1 paramutation and normal somatic development but without affecting overall small RNA biogenesis. In addition, genetic tests show this protein contributes to proper male gametophyte function. The similar mutant phenotypes documented in Arabidopsis and maize implicate some evolutionarily-conserved gene regulation while developmental defects associated with the two paramutation mutants are largely distinct. Our results show that a CHD3 protein responsible for normal plant ontogeny and sperm transmission also helps maintain meiotically-heritable epigenetic regulatory variation for specific alleles. This finding implicates an intersection of RNA polymerase IV function and nucleosome positioning in the paramutation process.

Deep sequencing of small non-coding RNA highlights brain-specific expression patterns and RNA cleavage.

With the advance of high-throughput sequencing technology numerous new regulatory small RNAs have been identified, that broaden the variety of processing mechanisms and functions of non-coding RNA. Here we explore small non-coding RNA (sncRNA) expression in central parts of the physiological stress and anxiety response system. Therefore, we characterize the sncRNA profile of tissue samples from Amygdala, Hippocampus, Hypothalamus and Adrenal Gland, obtained from 20 pigs. Our analysis reveals that all tissues but Amygdala and Hippocampus possess distinct, tissue-specific expression pattern of miRNA that are associated with Hypoxia, stress responses as well as memory and fear conditioning. In particular, we observe marked differences in the expression profile of limbic tissues compared to those associated to the HPA/stress axis, with a surprisingly high aggregation of 3´-tRNA halves in Amygdala and Hippocampus. Since regulation of sncRNA and RNA cleavage plays a pivotal role in the central nervous system, our work provides seminal insights in the role/involvement of sncRNA in the transcriptional and post-transcriptional regulation of negative emotion, stress and coping behaviour in pigs, and mammals in general.

Circulating small non-coding RNAs provide new insights into vitamin K nutrition and reproductive physiology in teleost fish.

BACKGROUND: Vitamin K (VK) is a fat-soluble vitamin known for its essential role in blood coagulation, but also on other biological processes (e.g. reproduction, brain and bone development) have been recently suggested. Nevertheless, the molecular mechanisms behind its particular function on reproduction are not yet fully understood. METHODS: The potential role of VK on reproduction through nutritional supplementation in Senegalese sole (Solea senegalensis) was assessed by gonadal maturation and 11-ketosterone, testosterone and estriol plasma levels when fed with control or VK supplemented (1250 mg kg-1 of VK1) diets along a six month trial. At the end, sperm production and quality (viability and DNA fragmentation) were evaluated. Circulating small non-coding RNAs (sncRNAs) in blood plasma from males were also studied through RNA-Seq. RESULTS: Fish fed with dietary VK supplementation had increased testosterone levels and lower sperm DNA fragmentation. SncRNAs from blood plasma were found differentially expressed when nutritional and sperm quality conditions were compared. PiR-675//676//4794//5462 and piR-74614 were found up-regulated in males fed with dietary VK supplementation. Let-7g, let-7e(18nt), let-7a-1, let-7a-3//7a-2//7a-1, let-7e(23nt) and piR-675//676//4794//5462 were found to be up-regulated and miR-146a and miR-146a-1//146a-2//146a-3 down-regulated when fish with low and high sperm DNA fragmentation were compared. Bioinformatic analyses of predicted mRNAs targeted by sncRNAs revealed the potential underlying pathways. CONCLUSIONS: VK supplementation improves fish gonad maturation and sperm quality, suggesting an unexpected and complex regulation of the nutritional status and reproductive performance through circulating sncRNAs. GENERAL SIGNIFICANCE: The use of circulating sncRNAs as reliable and less-invasive physiological biomarkers in fish nutrition and reproduction has been unveiled.

Identification of microRNA-like RNAs in Ophiocordyceps sinensis.

Ophiocordyceps sinensis is well known as a traditional Chinese medicine and has widely been used for over 2,000 years to stimulate immune system, decrease blood pressure and to inhibit tumor growth. While miRNAs are increasingly recognized for their roles in post-transcriptional regulation of gene expression in animals and plants, miRNAs in fungi were less studied until the discovery of microRNA-like RNA (milRNA). High-throughput sequencing and bioinformatics approaches were used to identify conserved and novel milRNAs in O. sinensis. 40 conserved milRNAs were identified, while 23 pre-miRNA candidates encoding 31 novel milRNAs were predicted. Furthermore, the potential target genes of milRNAs in human were predicted and gene ontology analysis was applied to these genes. Enrichment analysis of GO-represented biological process showed that target genes of both conserved and novel milRNAs are involved in development, metabolic and immune processes, indicating the potential roles of milRNAs of O. sinensis in pharmacological effects as health food and traditional Chinese medicine. This study is the first report on genome-wide analysis of milRNAs in O. sinensis and it provides a useful resource to further study the potential roles of milRNAs as active components of O. sinensis in health food or traditional Chinese medicine.

Large-scale analysis of small RNAs derived from traditional Chinese herbs in human tissues.

Plant-derived microRNAs have recently been reported to function in human blood and tissues. Controversy was immediately raised due to possible contamination and the lack of large sample sizes. Here, we report thousands of unique small RNAs derived from traditional Chinese medicine (TCM) herbs found in human blood cells and mouse lung tissues using a large-scale analysis. We extracted small RNAs from decoctions of 10 TCM plants (Ban Zhi Lian, Chai Hu, Chuan Xin Lian, Di Ding Zi Jin, Huang Qin, Jin Yin Hua, Lian Qiao, Pu Gong Ying, Xia Ku Cao, and Yu Xing Cao) and obtained millions of RNA sequences from each herb. We also obtained RNA-Seq data from the blood cells of humans who consumed herbal decoctions and from the lung tissues of mice administered RNAs from herbal decoctions via oral gavage. We identified thousands of unique small RNA sequences in human blood cells and mouse lung tissues. Some of these identified small RNAs from Chuan Xin Lian and Hong Jing Tian could be mapped to the genomes of the herbs, confirming their TCM plant origin. Small RNAs derived from herbs regulate mammalian gene expression in a sequence-specific manner, and thus are a superior novel class of herbal drug components that hold great potential as oral gene-targeted therapeutics, highlighting the important role of herbgenomics in their development.

Small RNA profiles from Panax notoginseng roots differing in sizes reveal correlation between miR156 abundances and root biomass levels.

Plant genomes encode several classes of small regulatory RNAs (sRNAs) that play critical roles in both development and stress responses. Panax notoginseng (Burk.) F.H. Chen (P. notoginseng) is an important traditional Chinese herbal medicinal plant species for its haemostatic effects. Therefore, the root yield of P. notoginseng is a major economically important trait since the roots of P. notoginseng are the parts used to produce medicine. To identify sRNAs that are critical for the root biomass of P. notoginseng, we performed a comprehensive study of miRNA transcriptomes from P. notoginseng roots of different biomasses. We identified 675 conserved miRNAs, of which 180 pre-miRNAs are also identified, and three TAS3 loci in P. notoginseng. By using degradome sequencing, we identified 79 conserved miRNA:target or tasiRNA:target interactions, of which eight were further confirmed with the RLM 5'-RACE experiments. More importantly, our results revealed that a member of miR156 family and one of its SPL target genes have inverse expression levels, which is tightly correlated with greater root biomass contents. These results not only contributes to overall understanding of post-transcriptional gene regulation in roots of P. notoginseng but also could serve as markers for breeding P. notoginseng with greater root yield.

Analysis of small RNAs revealed differential expressions during pollen and embryo sac development in autotetraploid rice.

BACKGROUND: Partial pollen and embryo sac sterilities are the two main reasons for low fertility in autotetraploid rice. Our previous study revealed that small RNAs changes may associate with pollen fertility in autotetraploid rice. However, knowledge on comparative analysis between the development of pollen and embryo sac by small RNAs in autotetraploid rice is still unknown. In the present study, WE-CLSM (whole-mount eosin B-staining confocal laser scanning microscopy) and high-throughput sequencing technology was employed to examine the cytological variations and to analyze small RNAs changes during pollen and embryo sac development in autotetraploid rice compared with its diploid counterpart. RESULTS: A total of 321 and 368 differentially expressed miRNAs (DEM) were detected during pollen and embryo sac development in autotetraploid rice, respectively. Gene Ontology enrichment analysis on the targets of DEM associated with embryo sac and pollen development revealed 30 prominent functional gene classes, such as cell differentiation and signal transduction during embryo sac development, while only 7 prominent functional gene classes, such as flower development and transcription factor activity, were detected during pollen development in autotetraploid rice. The expression levels of 39 DEM, which revealed interaction with meiosis-related genes, showed opposite expression patterns during pollen and embryo sac development. Of these DEM, osa-miR1436_L + 3_1ss5CT and osa-miR167h-3p were associated with the female meiosis, while osa-miR159a.1 and osa-MIR159a-p5 were related with the male meiosis. 21 nt-phasiRNAs were detected during both pollen and embryo sac development, while 24 nt-phasiRNAs were found only in pollen development, which displayed down-regulation in autotetraploid compared to diploid rice and their spatial-temporal expression patterns were similar to osa-miR2275d. 24 nt TEs-siRNAs were found to be up-regulated in embryo sac but down-regulated in pollen development. CONCLUSION: The above results not only provide the small RNAs changes during four landmark stages of pollen and embryo sac development in autotetraploid rice but also have identified specifically expressed miRNAs, especially meiosis-related miRNAs, pollen-specific-24 nt-phasiRNAs and TEs-siRNAs in autotetraploid rice. Together, these findings provide a foundation for understanding the effect of polyploidy on small RNAs expression patterns during pollen and embryo sac development that may lead to different abnormalities in autotetraploid rice.

Defining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery.

Small non-coding RNAs (ncRNA), including microRNAs (miRNA), enclosed in exosomes are being utilised for biomarker discovery in disease. Two common exosome isolation methods involve differential ultracentrifugation or differential ultracentrifugation coupled with Optiprep gradient fractionation. Generally, the incorporation of an Optiprep gradient provides better separation and increased purity of exosomes. The question of whether increased purity of exosomes is required for small ncRNA profiling, particularly in diagnostic and biomarker purposes, has not been addressed and highly debated. Utilizing an established neuronal cell system, we used next-generation sequencing to comprehensively profile ncRNA in cells and exosomes isolated by these 2 isolation methods. By comparing ncRNA content in exosomes from these two methods, we found that exosomes from both isolation methods were enriched with miRNAs and contained a diverse range of rRNA, small nuclear RNA, small nucleolar RNA and piwi-interacting RNA as compared with their cellular counterparts. Additionally, tRNA fragments (30-55 nucleotides in length) were identified in exosomes and may act as potential modulators for repressing protein translation. Overall, the outcome of this study confirms that ultracentrifugation-based method as a feasible approach to identify ncRNA biomarkers in exosomes.

Analysis of wheat microspore embryogenesis induction by transcriptome and small RNA sequencing using the highly responsive cultivar "Svilena".

BACKGROUND: Microspore embryogenesis describes a stress-induced reprogramming of immature male plant gametophytes to develop into embryo-like structures, which can be regenerated into doubled haploid plants after whole genome reduplication. This mechanism is of high interest for both research as well as plant breeding. The objective of this study was to characterize transcriptional changes and regulatory relationships in early stages of cold stress-induced wheat microspore embryogenesis by transcriptome and small RNA sequencing using a highly responsive cultivar. RESULTS: Transcriptome and small RNA sequencing was performed in a staged time-course to analyze wheat microspore embryogenesis induction. The analyzed stages were freshly harvested, untreated uninucleate microspores and the two following stages from in vitro anther culture: directly after induction by cold-stress treatment and microspores undergoing the first nuclear divisions. A de novo transcriptome assembly resulted in 29,388 contigs distributing to 20,224 putative transcripts of which 9,305 are not covered by public wheat cDNAs. Differentially expressed transcripts and small RNAs were identified for the stage transitions highlighting various processes as well as specific genes to be involved in microspore embryogenesis induction. CONCLUSION: This study establishes a comprehensive functional genomics resource for wheat microspore embryogenesis induction and initial understanding of molecular mechanisms involved. A large set of putative transcripts presumably specific for microspore embryogenesis induction as well as contributing processes and specific genes were identified. The results allow for a first insight in regulatory roles of small RNAs in the reprogramming of microspores towards an embryogenic cell fate.

The green tea polyphenol EGCG inhibits E. coli biofilm formation by impairing amyloid curli fibre assembly and downregulating the biofilm regulator CsgD via the σ(E) -dependent sRNA RybB.

In bacterial biofilms, which are often involved in chronic infections, cells are surrounded by a self-produced extracellular matrix that contains amyloid fibres, exopolysaccharides and other biopolymers. The matrix contributes to the pronounced resistance of biofilms against antibiotics and host immune systems. Being highly inflammatory, matrix amyloids such as curli fibres of Escherichia coli can also play a role in pathogenicity. Using macrocolony biofilms of commensal and pathogenic E. coli as a model system, we demonstrate here that the green tea polyphenol epigallocatachin gallate (EGCG) is a potent antibiofilm agent. EGCG virtually eliminates the biofilm matrix by directly interfering with the assembly of curli subunits into amyloid fibres, and by triggering the σ(E) cell envelope stress response and thereby reducing the expression of CsgD - a crucial activator of curli and cellulose biosynthesis - due to csgD mRNA targeting by the σ(E) -dependent sRNA RybB. These findings highlight EGCG as a potential adjuvant for antibiotic therapy of biofilm-associated infections. Moreover, EGCG may support therapies against pathogenic E. coli that produce inflammatory curli fibres along with Shigatoxin.

Identification of novel small ncRNAs in pollen of tomato.

BACKGROUND: The unprecedented role of sncRNAs in the regulation of pollen biogenesis on both transcriptional and epigenetic levels has been experimentally proven. However, little is known about their global regulation, especially under stress conditions. We used tomato pollen in order to identify pollen stage-specific sncRNAs and their target mRNAs. We further deployed elevated temperatures to discern stress responsive sncRNAs. For this purpose high throughput sncRNA-sequencing as well as Massive Analysis of cDNA Ends (MACE) were performed for three-replicated sncRNAs libraries derived from tomato tetrad, post-meiotic, and mature pollen under control and heat stress conditions. RESULTS: Using the omiRas analysis pipeline we identified known and predicted novel miRNAs as well as sncRNAs from other classes, responsive or not to heat. Differential expression analysis revealed that post-meiotic and mature pollen react most strongly by regulation of the expression of coding and non-coding genomic regions in response to heat. To gain insight to the function of these miRNAs, we predicted targets and annotated them to Gene Ontology terms. This approach revealed that most of them belong to protein binding, transcription, and Serine/Threonine kinase activity GO categories. Beside miRNAs, we observed differential expression of both tRNAs and snoRNAs in tetrad, post-meiotic, and mature pollen when comparing normal and heat stress conditions. CONCLUSIONS: Thus, we describe a global spectrum of sncRNAs expressed in pollen as well as unveiled those which are regulated at specific time-points during pollen biogenesis. We integrated the small RNAs into the regulatory network of tomato heat stress response in pollen.

Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing.

Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs.

MicL, a new σE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein.

In enteric bacteria, the transcription factor σ(E) maintains membrane homeostasis by inducing synthesis of proteins involved in membrane repair and two small regulatory RNAs (sRNAs) that down-regulate synthesis of abundant membrane porins. Here, we describe the discovery of a third σ(E)-dependent sRNA, MicL (mRNA-interfering complementary RNA regulator of Lpp), transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308-nucleotide (nt) primary transcript that is processed to an 80-nt form. Both forms possess features typical of Hfq-binding sRNAs but surprisingly target only a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that σ(E) activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new σ(E) regulatory loop that opposes membrane stress. Together MicA, RybB, and MicL allow σ(E) to repress the synthesis of all abundant outer membrane proteins in response to stress.

Comparative analysis of virus-specific small RNA profiles of three biologically distinct strains of Potato virus Y in infected potato (Solanum tuberosum) cv. Russet Burbank.

Deep sequencing technology has enabled the analysis of small RNA profiles of virus-infected plants and could provide insights into virus-host interactions. Potato virus Y is an economically important viral pathogen of potato worldwide. In this study, we investigated the nature and relative levels of virus-derived small interfering RNAs (vsiRNAs) in potato cv. Russet Burbank infected with three biologically distinct and economically important strains of PVY, the ordinary strain (PVY-O), tobacco veinal-necrotic strain (PVY-N) and tuber necrotic strain (PVY-NTN). The analysis showed an overall abundance of vsiRNAs of 20-24nt in PVY-infected plants. Considerable differences were present in the distribution of vsiRNAs as well as total small RNAs. The 21nt class was the most prevalent in PVY-infected plants irrespective of the virus strain, whereas in healthy potato plants, the 24nt class was the most dominant. vsiRNAs were derived from every position in the PVY genome, though certain hotspots were identified for each of the PVY strains. Among the three strains used, the population of vsiRNAs of different size classes was relatively different with PVY-NTN accumulating the highest level of vsiRNAs, while PVY-N infected plants had the least population of vsiRNAs. Unique vsiRNAs mapping to PVY genome in PVY-infected plants amounted to 3.13, 1.93 and 1.70% for NTN, N and O, respectively. There was a bias in the generation of vsiRNAs from the plus strand of the genome in comparison to the negative strand. The highest number of total vsiRNAs was from the cytoplasmic inclusion protein gene (CI) in PVY-O and PVY-NTN strains, whereas from PVY-N, the NIb gene produced maximum total vsiRNAs. These findings indicate that the three PVY strains interact differently in the same host genetic background and provided insights into virus-host interactions in an important food crop.