Regulatory mechanisms behind the phenotypic plasticity associated with Setaria italica water deficit tolerance.

Drought is one of the main environmental stresses that negatively impacts vegetative and reproductive yield. Water deficit responses are determined by the duration and intensity of the stress, which, together with plant genotype, will define the chances of plant survival. The metabolic adjustments in response to water deficit are complex and involve gene expression modulation regulated by DNA-binding proteins and epigenetic modifications. This last mechanism may also regulate the activity of transposable elements, which in turn impact the expression of nearby loci. Setaria italica plants submitted to five water deficit regimes were analyzed through a phenotypical approach, including growth, physiological, RNA-seq and sRNA-seq analyses. The results showed a progressive reduction in yield as a function of water deficit intensity associated with signaling pathway modulation and metabolic adjustments. We identified a group of loci that were consistently associated with drought responses, some of which were related to water deficit perception, signaling and regulation. Finally, an analysis of the transcriptome and sRNAome allowed us to identify genes putatively regulated by TE- and sRNA-related mechanisms and an intriguing positive correlation between transcript levels and sRNA accumulation in gene body regions. These findings shed light on the processes that allow S. italica to overcome drought and survive under water restrictive conditions.

Characterization and discovery of miRNA and miRNA targets from apomictic and sexual genotypes of Eragrostis curvula.

BACKGROUND: Weeping lovegrass (Eragrostis curvula [Shrad.] Nees) is a perennial grass found in semi-arid regions that is well adapted for growth in sandy soils and drought conditions. E. curvula constitutes a polymorphic complex that includes cytotypes with different ploidy levels (from 2x to 8x), where most polyploids are facultative apomicts, although both sexual reproduction and full apomixis have been reported in this species. Apomixis is thought to be associated with silencing of the sexual pathway, which would involve epigenetic mechanisms. However, a correlation between small RNAs and apomixis has not yet been conclusively established. RESULTS: Aiming to contribute to the elucidation of their role in the expression of apomixis, we constructed small RNA libraries from sexual and apomictic E. curvula genotypes via Illumina technology, characterized the small RNA populations, and conducted differential expression analysis by comparing these small RNAs with the E. curvula reference transcriptome. We found that the expression of two genes is repressed in the sexual genotype, which is associated with specific microRNA expression. CONCLUSION: Our results support the hypothesis that in E. curvula the expression of apomixis leads to sexual repression.

Identification of Hfq-binding RNAs in Caulobacter crescentus.

Small RNAs are important for post-transcriptional regulation of gene expression, affecting stability and activity of their target mRNAs. The bacterial Sm-like protein Hfq is required to promote pairing between both RNAs when their sequence complementarity is limited. To provide a first global view on the post-transcriptional landscape of the α-proteobacterium Caulobacter crescentus, we have identified the Hfq-binding RNAs employing High-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). A total of 261 RNAs, including 3 unannotated RNAs, were successfully identified and classified according to putative function. Moreover, possible interactions between the identified sRNAs with mRNA targets were postulated through computational target predictions.

An integrated analysis of mRNA and sRNA transcriptional profiles in Coffea arabica L. roots: insights on nitrogen starvation responses.

Coffea arabica L. is an important agricultural commodity, accounting for 60% of traded coffee worldwide. Nitrogen (N) is a macronutrient that is usually limiting to plant yield; however, molecular mechanisms of plant acclimation to N limitation remain largely unknown in tropical woody crops. In this study, we investigated the transcriptome of coffee roots under N starvation, analyzing poly-A+ libraries and small RNAs. We also evaluated the concentration of selected amino acids and N-source preferences in roots. Ammonium was preferentially taken up over nitrate, and asparagine and glutamate were the most abundant amino acids observed in coffee roots. We obtained 34,654 assembled contigs by mRNA sequencing, and validated the transcriptional profile of 12 genes by RT-qPCR. Illumina small RNA sequencing yielded 8,524,332 non-redundant reads, resulting in the identification of 86 microRNA families targeting 253 genes. The transcriptional pattern of eight miRNA families was also validated. To our knowledge, this is the first catalog of differentially regulated amino acids, N sources, mRNAs, and sRNAs in Arabica coffee roots.

Expression of the small regulatory RNA gene mmgR is regulated negatively by AniA and positively by NtrC in Sinorhizobium meliloti 2011.

In the N2-fixing symbiont of alfalfa root nodules, Sinorhizobium meliloti 2011, the mmgR gene encodes a 77 nt small untranslated RNA (sRNA) that negatively regulates the accumulation of polyhydroxybutyrate (PHB) when the bacterium is grown under conditions of surplus carbon (C) in relation to nitrogen (N). We previously showed that the expression of mmgR is primarily controlled at the transcriptional level and that it depends on the cellular N status, although the regulatory mechanism and the factors involved were unknown. In this study, we provide experimental data supporting that: (a) mmgR is induced upon N limitation with the maximum expression found at the highest tested C/N molar ratio in the growth medium; (b) a conserved heptamer TTGTGCA located between the -35 and -10 mmgR promoter elements is necessary and sufficient for induction by N limitation; (c) induction of mmgR requires the N-status regulator NtrC; (d) under C limitation, mmgR transcription is repressed by AniA, a global regulator of C flow; (e) the mmgR promoter contains a conserved dyadic motif (TGC[N3]GCA) partially overlapping the heptamer TTGTGCA, which was also found in the promoters of the PHB-related genes phaP1, phaP2, phaZ and phaR (aniA) of S. meliloti and other alpha-proteobacteria. Taken together, these results suggest that the mmgR promoter would integrate signals from the metabolism of C and N through - at least - the global regulators NtrC and AniA, to provide an optimal level of the MmgR sRNA to fine-tune gene expression post-transcriptionally according to varying C and N availability.

Virus-derived small RNAs: molecular footprints of host-pathogen interactions.

Viruses are obligatory intracellular parasites that require the host machinery to replicate. During their replication cycle, viral RNA intermediates can be recognized and degraded by different antiviral mechanisms that include RNA decay, RNA interference, and RNase L pathways. As a consequence of viral RNA degradation, infected cells can accumulate virus-derived small RNAs at high levels compared to cellular molecules. These small RNAs are imprinted with molecular characteristics that reflect their origin. First, small RNAs can be used to reconstruct viral sequences and identify the virus from which they originated. Second, other molecular features of small RNAs such as size, polarity, and base preferences depend on the type of viral substrate and host mechanism of degradation. Thus, the pattern of small RNAs generated in infected cells can be used as a molecular footprint to identify and characterize viruses independent on sequence homology searches against known references. Hence, sequencing of small RNAs obtained from infected cells enables virus discovery and characterization using both sequence-dependent strategies and novel pattern-based approaches. Recent studies are helping unlock the full application of small RNA sequencing for virus discovery and characterization. WIREs RNA 2016, 7:824-837. doi: 10.1002/wrna.1361 For further resources related to this article, please visit the WIREs website.

Phylogenetic distribution and evolutionary pattern of an α-proteobacterial small RNA gene that controls polyhydroxybutyrate accumulation in Sinorhizobium meliloti.

It has become clear that sRNAs play relevant regulatory functions in bacteria. However, a comprehensive understanding of their biological roles considering evolutionary aspects has not been achieved for most of them. Thus, we have characterized the evolutionary and phylogenetic aspects of the Sinorhizobium meliloti mmgR gene encoding the small RNA MmgR, which has been recently reported to be involved in the regulation of polyhydroxybutyrate accumulation in this bacterium. We constructed a covariance model from a multiple sequence and structure alignment of mmgR close homologs that allowed us to extend the search and to detect further remote homologs of the sRNA gene. From our results, mmgR seemed to evolve from a common ancestor of the α-proteobacteria that diverged from the order of Rickettsiales. We have found mmgR homologs in most current species of α-proteobacteria, with a few exceptions in which genomic reduction events or gene rearrangements seem to explain its absence. Furthermore, a strong microsyntenic relationship was found between a large set of mmgR homologs and homologs of a gene encoding a putative N-formyl glutamate amidohydrolase (NFGAH) that allowed us to trace back the evolutionary path of this group of mmgR orthologs. Among them, structure and sequence traits have been completely conserved throughout evolution, namely a Rho-independent terminator and a 10-mer (5'-UUUCCUCCCU-3') that is predicted to remain in a single-stranded region of the sRNA. We thus propose the definition of the new family of α-proteobacterial sRNAs αr8, as well as the subfamily αr8s1 which encompass S. meliloti mmgR orthologs physically linked with the downstream open reading frame encoding a putative NFGAH. So far, mmgR is the trans-encoded small RNA with the widest phylogenetic distribution of well recognized orthologs among α-proteobacteria. Expression of the expected MmgR transcript in rhizobiales other than S. meliloti (Sinorhizobium fredii, Rhizobium leguminosarum and Rhizobium etli) was confirmed by Northern blot. These findings will contribute to the understanding of the biological role(s) of mmgR in the α-proteobacteria.

RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond.

RegulonDB (http://regulondb.ccg.unam.mx) is one of the most useful and important resources on bacterial gene regulation,as it integrates the scattered scientific knowledge of the best-characterized organism, Escherichia coli K-12, in a database that organizes large amounts of data. Its electronic format enables researchers to compare their results with the legacy of previous knowledge and supports bioinformatics tools and model building. Here, we summarize our progress with RegulonDB since our last Nucleic Acids Research publication describing RegulonDB, in 2013. In addition to maintaining curation up-to-date, we report a collection of 232 interactions with small RNAs affecting 192 genes, and the complete repertoire of 189 Elementary Genetic Sensory-Response units (GENSOR units), integrating the signal, regulatory interactions, and metabolic pathways they govern. These additions represent major progress to a higher level of understanding of regulated processes. We have updated the computationally predicted transcription factors, which total 304 (184 with experimental evidence and 120 from computational predictions); we updated our position-weight matrices and have included tools for clustering them in evolutionary families. We describe our semiautomatic strategy to accelerate curation, including datasets from high-throughput experiments, a novel coexpression distance to search for 'neighborhood' genes to known operons and regulons, and computational developments.

Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines.

Intercellular communication can be mediated by extracellular small regulatory RNAs (sRNAs). Circulating sRNAs are being intensively studied for their promising use as minimally invasive disease biomarkers. To date, most attention is centered on exosomes and microRNAs as the vectors and the secreted species, respectively. However, this field would benefit from an increased understanding of the plethora of sRNAs secreted by different cell types in different extracellular fractions. It is still not clear if specific sRNAs are selected for secretion, or if sRNA secretion is mostly passive. We sequenced the intracellular sRNA content (19-60 nt) of breast epithelial cell lines (MCF-7 and MCF-10A) and compared it with extracellular fractions enriched in microvesicles, exosomes and ribonucleoprotein complexes. Our results are consistent with a non-selective secretion model for most microRNAs, although a few showed secretion patterns consistent with preferential secretion. On the contrary, 5' tRNA halves and 5' RNA Y4-derived fragments of 31-33 were greatly and significantly enriched in the extracellular space (even in non-mammary cell lines), where tRNA halves were detected as part of ∼45 kDa ribonucleoprotein complexes. Overall, we show that different sRNA families have characteristic secretion patterns and open the question of the role of these sRNAs in the extracellular space.