siRNA Clusters Research Articles

Dynamic roles of small RNAs and DNA methylation associated with heterosis in allotetraploid cotton (Gossypium hirsutum L.)

BackgroundHeterosis is a complex phenomenon wherein the hybrids outperform their parents. Understanding the underlying molecular mechanism by which hybridization leads to higher yields in allopolyploid cotton is critical for effective breeding programs. Here, we integrated DNA methylation, transcriptomes, and small RNA profiles to comprehend the genetic and molecular basis of heterosis in allopolyploid cotton at three developmental stages.ResultsTranscriptome analysis revealed that numerous DEGs responsive to phytohormones (auxin and salicylic acid) were drastically altered in F1 hybrid compared to the parental lines. DEGs involved in energy metabolism and plant growth were upregulated, whereas DEGs related to basal defense were downregulated. Differences in homoeologous gene expression in F1 hybrid were greatly reduced after hybridization, suggesting that higher levels of parental expression have a vital role in heterosis. Small RNAome and methylome studies showed that the degree of DNA methylation in hybrid is higher when compared to the parents. A substantial number of allele-specific expression genes were found to be strongly regulated by CG allele-specific methylation levels. The hybrid exhibited higher 24-nt-small RNA (siRNA) expression levels than the parents. The regions in the genome with increased levels of 24-nt-siRNA were chiefly related to genes and their flanking regulatory regions, demonstrating a possible effect of these molecules on gene expression. The transposable elements correlated with siRNA clusters in the F1 hybrid had higher methylation levels but lower expression levels, which suggest that these non-additively expressed siRNA clusters, reduced the activity of transposable elements through DNA methylation in the hybrid.ConclusionsThese multi-omics data provide insights into how changes in epigenetic mechanisms and gene expression patterns can lead to heterosis in allopolyploid cotton. This makes heterosis a viable tool in cotton breeding.

Integrated Multi-Omics Reveals Significant Roles of Non-Additively Expressed Small RNAs in Heterosis for Maize Plant Height.

Heterosis is a complex biological phenomenon regulated by genetic variations and epigenetic changes. However, the roles of small RNAs (sRNAs), an important epigenetic regulatory element, on plant heterosis are still poorly understood. Here, an integrative analysis was performed with sequencing data from multi-omics layers of maize hybrids and their two homologous parental lines to explore the potential underlying mechanisms of sRNAs in plant height (PH) heterosis. sRNAome analysis revealed that 59 (18.61%) microRNAs (miRNAs) and 64,534 (54.00%) 24-nt small interfering RNAs (siRNAs) clusters were non-additively expressed in hybrids. Transcriptome profiles showed that these non-additively expressed miRNAs regulated PH heterosis through activating genes involved in vegetative growth-related pathways while suppressing those related to reproductive and stress response pathways. DNA methylome profiles showed that non-additive methylation events were more likely to be induced by non-additively expressed siRNA clusters. Genes associated with low-parental expression (LPE) siRNAs and trans-chromosomal demethylation (TCdM) events were enriched in developmental processes as well as nutrients and energy metabolism, whereas genes associated with high-parental expression (HPE) siRNAs and trans-chromosomal methylation (TCM) events were gathered in stress response and organelle organization pathways. Our results provide insights into the expression and regulation patterns of sRNAs in hybrids and help to elucidate their potential targeting pathways contributing to PH heterosis.

The Rise and Fall of Billionaire siRNAs during Reproductive Development in Rice.

RNA polymerase IV-dependent siRNAs, usually 24 nt in length, function in the RNA-directed DNA methylation that is responsible for de novo methylation in plants. We analyzed 24 nt siRNAs in inflorescences and found that among the 20,200 24 nt siRNA clusters, the top 0.81% highly expressed clusters accounted for more than 68% of the 24 nt siRNA reads in inflorescences. We named the highly expressed siRNAs as billionaire siRNAs (bill-siRNAs) and the less-expressed siRNAs as pauper siRNAs (pau-siRNAs). The bill-siRNAs in inflorescences are mainly derived from the ovary. Female gametes produced more bill-siRNAs than male gametes. In embryos and seedlings developed from fertilized egg cells, the bill-siRNAs from gametes disappeared. The endosperm, which develops from the fertilized central cell, also contained no bill-siRNAs from gametes but did contain newly and highly expressed siRNAs produced in different regions. In contrast, bill-siRNAs from the ovaries were maintained in the seed coat. The biosynthesis of bill-siRNAs in various tissues and cells is dependent on OsRDR2 (RNA-dependent RNA polymerase 2) and Pol IV (DNA-dependent RNA polymerase IV). Similar to the pau-siRNAs, the first base of bill-siRNAs is enriched at adenine, and bill-siRNAs can direct DNA methylation in various tissues.

Transcriptional regulatory networks in response to drought stress and rewatering in maize (Zea mays L.).

Drought severely affects the growth and development of maize, but there is a certain degree of compensation effect after rewatering. This study intends to elaborate the response mechanism of maize at the physiological and molecular level as well as excavating potential genes with strong drought resistance and recovery ability. Physiological indexes analysis demonstrated that stomata conductance, transpiration rate, photosynthesis rate, antioxidant enzymes, and proline levels in maize were significantly altered in response to drought for 60 and 96h and rewatering for 3days. At 60h, 96h, and R3d, we detected 3095, 1941, and 5966 differentially expressed genes (DEGs) and 221, 226, and 215 differentially expressed miRNAs. Weighted correlation network analysis (WGCNA) showed that DEGs responded to maize drought and rewatering through participating in photosynthesis, proline metabolism, ABA signaling, and oxidative stress. Joint analysis of DEGs, miRNA, and target genes showed that zma-miR529, miR5072, zma-miR167e, zma-miR167f, zma-miR167j, miR397, and miR6214 were involved to regulate SBPs, MYBs, ARFs, laccases, and antioxidant enzymes, respectively. Hundreds of differentially expressed DNA methylation-related 24-nt siRNA clusters overlap with DEGs, indicating that DNA methylation is involved in responses under drought stress. These results provide new insights into the molecular mechanisms of drought tolerance, and may identify new targets for breeding drought-tolerant maize lines.

Genome‐wide analysis of epigenetic and transcriptional changes associated with heterosis in pigeonpea

SummaryHybrids are extensively used in agriculture to deliver an increase in yield, yet the molecular basis of heterosis is not well understood. Global DNA methylation analysis, transcriptome analysis and small RNA profiling were aimed to understand the epigenetic effect of the changes in gene expression level in the two hybrids and their parental lines. Increased DNA methylation was observed in both the hybrids as compared to their parents. This increased DNA methylation in hybrids showed that majority of the 24‐nt siRNA clusters had higher expression in hybrids than the parents. Transcriptome analysis revealed that various phytohormones (auxin and salicylic acid) responsive hybrid‐MPV DEGs were significantly altered in both the hybrids in comparison to MPV. DEGs associated with plant immunity and growth were overexpressed whereas DEGs associated with basal defence level were repressed. This antagonistic patterns of gene expression might contribute to the greater growth of the hybrids. It was also noticed that some common as well as unique changes in the regulatory pathways were associated with heterotic growth in both the hybrids. Approximately 70% and 67% of down‐regulated hybrid‐MPV DEGs were found to be differentially methylated in ICPH 2671 and ICPH 2740 hybrid, respectively. This reflected the association of epigenetic regulation in altered gene expressions. Our findings also revealed that miRNAs might play important roles in hybrid vigour in both the hybrids by regulating their target genes, especially in controlling plant growth and development, defence and stress response pathways. The above finding provides an insight into the molecular mechanism of pigeonpea heterosis.

Exogenous RNAi mechanisms contribute to transcriptome adaptation by phased siRNA clusters in Paramecium.

Extensive research has characterized distinct exogenous RNAi pathways interfering in gene expression during vegetative growth of the unicellular model ciliate Paramecium. However, role of RNAi in endogenous transcriptome regulation, and environmental adaptation is unknown. Here, we describe the first genome-wide profiling of endogenous sRNAs in context of different transcriptomic states (serotypes). We developed a pipeline to identify, and characterize 2602 siRNA producing clusters (SRCs). Our data show no evidence that SRCs produce miRNAs, and in contrast to other species, no preference for strand specificity of siRNAs. Interestingly, most SRCs overlap coding genes and a separate group show siRNA phasing along the entire open reading frame, suggesting that the mRNA transcript serves as a source for siRNAs. Integrative analysis of siRNA abundance and gene expression levels revealed surprisingly that mRNA and siRNA show negative as well as positive associations. Two RNA-dependent RNA Polymerase mutants, RDR1 and RDR2, show a drastic loss of siRNAs especially in phased SRCs accompanied with increased mRNA levels. Importantly, most SRCs depend on both RDRs, reminiscent to primary siRNAs in the RNAi against exogenous RNA, indicating mechanistic overlaps between exogenous and endogenous RNAi contributing to flexible transcriptome adaptation.

Characterization of siRNAs clusters in Arabidopsis thaliana galls induced by the root-knot nematode Meloidogyne incognita

BackgroundRoot-knot nematodes (RKN), genus Meloidogyne, are plant parasitic worms that have the ability to transform root vascular cylinder cells into hypertrophied, multinucleate and metabolically over-active feeding cells. Redifferentiation into feeding cells is the result of a massive transcriptional reprogramming of root cells targeted by RKN. Since RKN are able to induce similar feeding cells in roots of thousands of plant species, these worms are thought to manipulate essential and conserved plant molecular pathways.ResultsSmall non-coding RNAs of uninfected roots and infected root galls induced by M. incognita from Arabidopsis thaliana were sequenced by high throughput sequencing. SiRNA populations were analysed by using the Shortstack algorithm. We identified siRNA clusters that are differentially expressed in infected roots and evidenced an over-representation of the 23–24 nt siRNAs in infected tissue. This size corresponds to heterochromatic siRNAs (hc-siRNAs) which are known to regulate expression of transposons and genes at the transcriptional level, mainly by inducing DNA methylation.ConclusionsCorrelation of siRNA clusters expression profile with transcriptomic data identified several protein coding genes that are candidates to be regulated by siRNAs at the transcriptional level by RNA directed DNA methylation (RdDM) pathway either directly or indirectly via silencing of neighbouring transposable elements.

Analysis of transcriptional and epigenetic changes in hybrid vigor of allopolyploid Brassica napus uncovers key roles for small RNAs.

Heterosis is a fundamental biological phenomenon characterized by the superior performance of a hybrid compared with its parents. The underlying molecular basis for heterosis, particularly for allopolyploids, remains elusive. In this study we analyzed the transcriptomes of Brassica napus parental lines and their F1 hybrids at three stages of early flower development. Phenotypically, the F1 hybrids show remarkable heterosis in silique number and grain yield. Transcriptome analysis revealed that various phytohormone (auxin and salicylic acid) response genes are significantly altered in the F1 hybrids relative to the parental lines. We also found evidence for decreased expression divergence of the homoeologous gene pairs in the allopolyploid F1 hybrids and suggest that high-parental expression-level dominance plays an important role in heterosis. Small RNA and methylation studies aimed at examining the epigenetic effect of the changes in gene expression level in the F1 hybrids showed that the majority of the small interfering RNA (siRNA) clusters had a higher expression level in the F1 hybrids than in the parents, and that there was an increase in genome-wide DNA methylation in the F1 hybrid. Transposable elements associated with siRNA clusters had a higher level of methylation and a lower expression level in the F1 hybrid, implying that the non-additively expressed siRNA clusters resulted in lower activity of the transposable elements through DNA methylation in the hybrid. Our data provide insights into the role that changes in gene expression pattern and epigenetic mechanisms contribute to heterosis during early flower development in allopolyploid B.napus.

Clustering and evolutionary analysis of small RNAs identify regulatory siRNA clusters induced under drought stress in rice.

MotivationDrought tolerance is an important trait related to growth and yield in crop. Until now, drought related research has focused on coding genes. However, non-coding RNAs also respond significantly to environmental stimuli such as drought stress. Unfortunately, characterizing the role of siRNAs under drought stress is difficult since a large number of heterogenous siRNA species are expressed under drought stress and non-coding RNAs have very weak evolutionary conservation. Thus, to characterize the role of siRNAs, we need a well designed biological and bioinformatics strategy. In this paper, to characterize the function of siRNAs we developed and used a bioinformatics pipeline that includes a genomic-location based clustering technique and an evolutionary conservation tool.ResultsBy comparing the wild type Nipponbare and two drought resistant rice varities, we found that 21 nt and 24 nt siRNAs are significantly expressed in the three rice plants but at different time points under a short-term (0, 1, and 6 hrs) drought treatment. siRNAs were up-regulated in the wild type at an early stage while the up-regulation was delayed in the two drought tolerant plants. Genes targeted by up-regulated siRNAs were related to oxidation reduction and proteolysis, which are well known to be associated with water deficit phenotypes. More interestingly, we found that siRNAs were located in intronic regions as clusters and were of high evolutionary conservation among monocot grass plants. In summary, we show that siRNAs are important respondents to drought stress and regulate genes related to the drought tolerance in water deficit conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0355-3) contains supplementary material, which is available to authorized users.

Genome-wide analysis of single non-templated nucleotides in plant endogenous siRNAs and miRNAs.

Plant small RNAs are subject to various modifications. Previous reports revealed widespread 3′ modifications (truncations and non-templated tailing) of plant miRNAs when the 2′-O-methyltransferase HEN1 is absent. However, non-templated nucleotides in plant heterochromatic siRNAs have not been deeply studied, especially in wild-type plants. We systematically studied non-templated nucleotide patterns in plant small RNAs by analyzing small RNA sequencing libraries from Arabidopsis, tomato, Medicago, rice, maize and Physcomitrella. Elevated rates of non-templated nucleotides were observed at the 3′ ends of both miRNAs and endogenous siRNAs from wild-type specimens of all species. ‘Off-sized’ small RNAs, such as 25 and 23 nt siRNAs arising from loci dominated by 24 nt siRNAs, often had very high rates of 3′-non-templated nucleotides. The same pattern was observed in all species that we studied. Further analysis of 24 nt siRNA clusters in Arabidopsis revealed distinct patterns of 3′-non-templated nucleotides of 23 nt siRNAs arising from heterochromatic siRNA loci. This pattern of non-templated 3′ nucleotides on 23 nt siRNAs is not affected by loss of known small RNA 3′-end modifying enzymes, and may result from modifications added to longer heterochromatic siRNA precursors.

Clustering siRNA conjugates for MMP-responsive therapeutics in chronic wounds of diabetic animals.

The MMP-responsive breakdown of siRNA clusters was translated to site-specific gene transfection and enhanced wound healing in diabetic ulcers. MMP-2 siRNA was chemically tethered to the end of multi-armed PEG via MMP-cleavable linkers (4PEG-siRNA) and subsequently clustered into submicron particles complexed with LPEI. 4PEG-siRNA was more tightly complexed with LPEI and the associated cluster showed higher resistance against RNase attack, in comparison to naked siRNA. Because the size of the clusters increased depending on the increase in charge ratio of LPEI to siRNA, cellular uptake of the 4PEG-siRNA/LPEI cluster was significantly attenuated due to the huge size of the cluster. However, upon MMP treatment, the cluster dissociated into smaller particles and was efficiently endocytosed by cells. An in vivo fluorescence resonance energy transfer (FRET) study also revealed that the clusters were effectively dissociated in MMP-rich environments of dorsal wounds in diabetic animals. In addition, diabetic ulcers treated with the clusters showed a faster wound closure rate and the recovered tissue expressed a larger amount of cytokeratin along with a lower expression level of MMP-2 compared to the other groups.

Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge.

Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S)lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.

Characterization and expression patterns of small RNAs in synthesized Brassica hexaploids

Polyploidy has played an important role in promoting plant evolution through genomic merging and doubling. We used high-throughput sequencing to compare miRNA expression profiles between Brassica hexaploid and its parents. A total of 613, 784 and 742 known miRNAs were identified in Brassica rapa, Brassica carinata, and Brassica hexaploid, respectively. We detected 618 miRNAs were differentially expressed (log(2)Ratio ≥ 1, P ≤ 0.05) between Brassica hexaploid and its parents, and 425 miRNAs were non-additively expressed in Brassica hexaploid, which suggest a trend of non-additive miRNA regulation following hybridization and polyploidization. Remarkably, majority of the non-additively expressed miRNAs in the Brassica hexaploid are repressed, and there was a bias toward repression of B. rapa miRNAs, which is consistent with the progenitor-biased gene repression in the synthetic allopolyploids. In addition, we identified 653 novel mature miRNAs in Brassica hexaploid and its parents. Finally, we found that almost all the non-additive accumulation of siRNA clusters exhibited a low-parent pattern in Brassica hexaploid. Non-additive small RNA regulation is involved in a range of biological pathways, probably providing a driving force for variation and adaptation in allopolyploids.

Epigenetic genome control by heterochromatin and RNAi machinery

RNAi is a conserved mechanism in which small interfering RNAs (siRNAs) guide the degradation of cognate RNAs, but also promote heterochromatin assembly at repetitive DNA elements such as centromeric repeats. However, the full extent of RNAi functions and its endogenous targets have not been explored. We show that in the fission yeast Schizosaccharomyces pombe, RNAi and heterochromatin factors cooperate to silence diverse loci, including sexual differentiation genes, genes encoding transmembrane proteins, and retrotransposons that are also targeted by the exosome RNA degradation machinery. In the absence of the exosome, transcripts are processed preferentially by the RNAi, revealing siRNA clusters and corresponding increase in heterochromatin modifications across large domains containing genes and retrotransposons. Interestingly, the generation of siRNA clusters and heterochromatin assembly by RNAi is triggered by a mechanism involving the canonical poly(A) polymerase Pla1 and an associated RNA surveillance factor Red1, which also activate the exosome. More importantly, siRNA production and heterochromatin modifications at these target loci are regulated by environmental growth conditions, and by developmental signals that induce gene expression during sexual differentiation. These analyses uncover interplay between RNAi and the exosome that is conserved in higher eukaryotes, and show that differentiation signals modulate RNAi silencing to regulate developmental genes.

RNAi triggered by specialized machinery silences developmental genes and retrotransposons

RNAi is a conserved mechanism in which small interfering RNAs (siRNAs) guide the degradation of cognate RNAs, but also promote heterochromatin assembly at repetitive DNA elements such as centromeric repeats1,2. However, the full extent of RNAi functions and its endogenous targets have not been explored. Here we show that in the fission yeast Schizosaccharomyces pombe, RNAi and heterochromatin factors cooperate to silence diverse loci, including sexual differentiation genes, genes encoding transmembrane proteins, and retrotransposons that are also targeted by the exosome RNA degradation machinery. In the absence of the exosome, transcripts are processed preferentially by the RNAi machinery, revealing siRNA clusters and corresponding increase in heterochromatin modifications across large domains containing genes and retrotransposons. We show that the generation of siRNAs and heterochromatin assembly by RNAi is triggered by a mechanism involving the canonical poly(A) polymerase Pla1 and an associated RNA surveillance factor Red1, which also activate the exosome. Remarkably, siRNA production and heterochromatin modifications at these target loci are regulated by environmental growth conditions, and by developmental signals that induce gene expression during sexual differentiation. Our analyses uncover interplay between RNAi and the exosome that is conserved in higher eukaryotes, and show that differentiation signals modulate RNAi silencing to regulate developmental genes.

The Inheritance Pattern of 24 nt siRNA Clusters in Arabidopsis Hybrids Is Influenced by Proximity to Transposable Elements

Hybrids often display increased size and growth, and thus are widely cultivated in agriculture and horticulture. Recent discoveries demonstrating the important regulatory roles of small RNAs have greatly improved our understanding of many basic biological questions, and could illuminate the molecular basis for the enhanced growth and size of hybrid plants. We profiled small RNAs by deep sequencing to characterize the inheritance patterns of small RNA levels in reciprocal hybrids of two Arabidopsis thaliana accessions, Columbia and Landsberg erecta. We find 24-nt siRNAs predominate among those small RNAs that are differentially expressed between the parents. Following hybridization, the transposable element (TE)-derived siRNAs are often inherited in an additive manner, whereas siRNAs associated with protein-coding genes are often down-regulated in hybrids to the levels observed for the parent with lower relative siRNA levels. Among the protein-coding genes that exhibit this pattern, genes that function in pathogen defense, abiotic stress tolerance, and secondary metabolism are significantly enriched. Small RNA clusters from protein-coding genes where a TE is present within one kilobase show a different predominant inheritance pattern (additive) from those that do not (low-parent dominance). Thus, down-regulation in the form of low-parent dominance is likely the default pattern of inheritance for genic siRNA, and a different inheritance mechanism for TE siRNA is suggested.

Tandem repeats upstream of the Arabidopsis endogene SDC recruit non-CG DNA methylation and initiate siRNA spreading

Plants use siRNAs to target cytosine DNA methylation to both symmetrical CG and nonsymmetrical (CHG and CHH) sequence contexts. DNA methylation and siRNA clusters most frequently overlap with transposons in the Arabidopsis thaliana genome. However, a significant number of protein-coding genes also show promoter DNA methylation, and this can be used to silence their expression. Loss of the majority of non-CG DNA methylation in drm1 drm2 cmt3 triple mutants leads to developmental phenotypes. We identified the gene responsible for these phenotypes as SUPPRESSOR OF drm1 drm2 cmt3 (SDC), which encodes an F-box protein and possesses seven promoter tandem repeats. The SDC repeats show a unique silencing requirement for non-CG DNA methylation directed redundantly by histone methylation and siRNAs, and display spreading of siRNAs and methylation beyond the repeated region. In addition to revealing the complexity of DNA methylation control in A. thaliana, SDC has important implications for how plant genomes utilize gene silencing to repress endogenous genes.

Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster

Cis-natural antisense transcripts (cis-NATs) have been speculated to be substrates for endogenous RNA interference (RNAi), but little experimental evidence for such a pathway in animals has been reported. Analysis of massive Drosophila melanogaster small RNA data sets now reveals two mechanisms that yield endogenous small interfering RNAs (siRNAs) via bidirectional transcription. First, >100 cis-NATs with overlapping 3' exons generate 21-nt, and, based on previously published small RNA data [corrected] Dicer-2 (Dcr-2)-dependent, 3'-end modified siRNAs. The processing of cis-NATs by RNA interference (RNAi) seems to be actively restricted, and the selected loci are enriched for nucleic acid-based functions and include Argonaute-2 (AGO2) itself. Second, we report that extended intervals of the thickveins and klarsicht genes generate exceptionally abundant siRNAs from both strands. These siRNA clusters derive from atypical cis-NAT arrangements involving introns and 5' or internal exons, but their biogenesis is similarly Dcr-2- and AGO2-dependent. These newly recognized siRNA pathways broaden the scope of regulatory networks mediated by small RNAs.