Deciphering shared attributes of plant long non-coding RNAs through a comparative computational approach.

Over the past decade, long non-coding RNA (lncRNA), which lacks protein-coding potential, has emerged as an essential regulator of the genome. The present study examined 13,599 lncRNAs in Arabidopsis thaliana, 11,565 in Oryza sativa, and 32,397 in Zea mays for their characteristic features and explored the associated genomic and epigenomic features. We found lncRNAs were distributed throughout the chromosomes and the Helitron family of transposable elements (TEs) enriched, while the terminal inverted repeat depleted in lncRNA transcribing regions. Our analyses determined that lncRNA transcribing regions show rare or weak signals for most epigenetic marks except for H3K9me2 and cytosine methylation in all three plant species. LncRNAs showed preferential localization in the nucleus and cytoplasm; however, the distribution ratio in the cytoplasm and nucleus varies among the studied plant species. We identified several conserved endogenous target mimic sites in the lncRNAs among the studied plants. We found 233, 301, and 273 unique miRNAs, potentially targeting the lncRNAs of A. thaliana, O. sativa, and Z. mays, respectively. Our study has revealed that miRNAs, which interact with lncRNAs, target genes that are involved in a diverse array of biological and molecular processes. The miRNA-targeted lncRNAs displayed a strong affinity for several transcription factors, including ERF and BBR-BPC, mutually present in all three plants, advocating their conserved functions. Overall, the present study showed that plant lncRNAs exhibit conserved genomic and epigenomic characteristics and potentially govern the growth and development of plants.

Genome-wide analysis of long non-coding RNAs under diel light exhibits role in floral development and the circadian clock in Arabidopsis thaliana.

The circadian clock is regulated by signaling networks that enhance a plant's ability to coordinate internal events with the external environment. In this study, we examine the rhythmic expression of long non-coding RNAs (lncRNAs) using multiple transcriptomes of Arabidopsis thaliana in the diel light cycle and integrated this information to have a better understanding of the functions of lncRNAs in regulating the circadian clock. We identified 968, 1050, and 998 lncRNAs at 8 h light, 16 h light and 8 h dark conditions, respectively. Among these, 423, 486, and 417 lncRNAs were uniquely present at 8 h light, 16 h light, and 8 h dark, respectively, whereas 334 lncRNAs were common under the three conditions. The specificity of identified lncRNAs under different light conditions was verified using qRT-PCR. The identified lncRNAs were less GC-rich and expressed at a significantly lower level than the mRNAs of protein-coding genes. In addition, we identified enriched motifs in lncRNA transcribing regions that were associated with light-responsive genes (SORLREP and SORLIP), flower development (AGAMOUS), and circadian clock (CCA1) under all three light conditions. We identified 10 and 12 different lncRNAs targeting different miRNAs with perfect and interrupted complementarity (endogenous target mimic). These predicted lncRNA-interacting miRNAs govern the function of a set of genes involved in the developmental process, reproductive structure development, gene silencing and transcription regulation. We demonstrated that the lncRNA transcribing regions were enriched for epigenetic marks such as H3.3, H3K4me2, H3K4me3, H4K16ac, H3K36ac, H3K56ac and depleted for heterochromatic (H3K9me2 and H3K27me1) and repressive (H3K27me3) histone modifications. Further, we found that hypermethylated genomic regions negatively correlated with lncRNA transcribing regions. Overall, our study showed that lncRNAs expressed corresponding to the diel light cycle are implicated in regulating the circadian rhythm and governing the developmental stage-specific growth.

Mechanism and Control of Meiotic DNA Double-Strand Break Formation in S. cerevisiae.

Developmentally programmed formation of DNA double-strand breaks (DSBs) by Spo11 initiates a recombination mechanism that promotes synapsis and the subsequent segregation of homologous chromosomes during meiosis. Although DSBs are induced to high levels in meiosis, their formation and repair are tightly regulated to minimize potentially dangerous consequences for genomic integrity. In S. cerevisiae, nine proteins participate with Spo11 in DSB formation, but their molecular functions have been challenging to define. Here, we describe our current view of the mechanism of meiotic DSB formation based on recent advances in the characterization of the structure and function of DSB proteins and discuss regulatory pathways in the light of recent models.

INT-Hi-C reveals distinct chromatin architecture in endosperm and leaf tissues of Arabidopsis.

Higher-order chromatin structure undergoes striking changes in response to various developmental and environmental signals, causing distinct cell types to adopt specific chromatin organization. High throughput chromatin conformation capture (Hi-C) allows studying higher-order chromatin structure; however, this technique requires substantial amounts of starting material, which has limited the establishment of cell type-specific higher-order chromatin structure in plants. To overcome this limitation, we established a protocol that is applicable to a limited amount of nuclei by combining the INTACT (isolation of nuclei tagged in specific cell types) method and Hi-C (INT-Hi-C). Using this INT-Hi-C protocol, we generated Hi-C data from INTACT purified endosperm and leaf nuclei. Our INT-Hi-C data from leaf accurately reiterated chromatin interaction patterns derived from conventional leaf Hi-C data. We found that the higher-order chromatin organization of mixed leaf tissues and endosperm differs and that DNA methylation and repressive histone marks positively correlate with the chromatin compaction level. We furthermore found that self-looped interacting genes have increased expression in leaves and endosperm and that interacting intergenic regions negatively impact on gene expression in the endosperm. Last, we identified several imprinted genes involved in long-range and trans interactions exclusively in endosperm. Our study provides evidence that the endosperm adopts a distinct higher-order chromatin structure that differs from other cell types in plants and that chromatin interactions influence transcriptional activity.

Stress Conditions Modulate the Chromatin Interactions Network in Arabidopsis.

Stresses have been known to cause various responses like cellular physiology, gene regulation, and genome remodeling in the organism to cope and survive. Here, we assessed the impact of stress conditions on the chromatin-interactome network of Arabidopsis thaliana. We identified thousands of chromatin interactions in native as well as in salicylic acid treatment and high temperature conditions in a genome-wide fashion. Our analysis revealed the definite pattern of chromatin interactions and stress conditions could modulate the dynamics of chromatin interactions. We found the heterochromatic region of the genome actively involved in the chromatin interactions. We further observed that the establishment or loss of interactions in response to stress does not result in the global change in the expression profile of interacting genes; however, interacting regions (genes) containing motifs for known TFs showed either lower expression or no difference than non-interacting genes. The present study also revealed that interactions preferred among the same epigenetic state (ES) suggest interactions clustered the same ES together in the 3D space of the nucleus. Our analysis showed that stress conditions affect the dynamics of chromatin interactions among the chromatin loci and these interaction networks govern the folding principle of chromatin by bringing together similar epigenetic marks.

Correction to: Epigenetic signatures associated with imprinted paternally expressed genes in the Arabidopsis endosperm.

Following publication of the original article [1], the authors reported that Additional file 4, "Table S5. Parent-of-origin RNAseq dataset of 4 DAP INTACT-purified endosperm of Col × Ler reciprocal crosses" had the following error.

Epigenetic signatures associated with imprinted paternally expressed genes in the Arabidopsis endosperm.

BACKGROUND: Imprinted genes are epigenetically modified during gametogenesis and maintain the established epigenetic signatures after fertilization, causing parental-specific gene expression. RESULTS: In this study, we show that imprinted paternally expressed genes (PEGs) in the Arabidopsis endosperm are marked by an epigenetic signature of Polycomb Repressive Complex2 (PRC2)-mediated H3K27me3 together with heterochromatic H3K9me2 and CHG methylation, which specifically mark the silenced maternal alleles of PEGs. The co-occurrence of H3K27me3 and H3K9me2 on defined loci in the endosperm drastically differs from the strict separation of both pathways in vegetative tissues, revealing tissue-specific employment of repressive epigenetic pathways in plants. Based on the presence of this epigenetic signature on maternal alleles, we are able to predict known PEGs at high accuracy and identify several new PEGs that we confirm using INTACT-based transcriptomes generated in this study. CONCLUSIONS: The presence of the three repressive epigenetic marks, H3K27me3, H3K9me2, and CHG methylation on the maternal alleles in the endosperm serves as a specific epigenetic signature that allows prediction of genes with parental-specific gene expression. Our study reveals that there are substantially more PEGs than previously identified, indicating that paternal-specific gene expression is of higher functional relevance than currently estimated. The combined activity of PRC2-mediated H3K27me3 together with the heterochromatic H3K9me3 has also been reported to silence the maternal Xist locus in mammalian preimplantation embryos, suggesting convergent employment of both pathways during the evolution of genomic imprinting.

GhMYB1 regulates SCW stage-specific expression of the GhGDSL promoter in the fibres of Gossypium hirsutum L.

Secondary cell wall (SCW) biosynthesis is an important stage of the cotton fibre development, and its transcriptional regulation is poorly understood. We selected the Gossypium hirsutum GDSL (GhGDSL) lipase/hydrolase gene (CotAD_74480), which is expressed during SCW biosynthesis (19 through to 25 days postanthesis; DPA), for study. T1 -transgenic cotton lines expressing the ß-glucuronidase (gus) reporter under the control of a 1026-bp promoter fragment of GhGDSL (PGhGDSL ) showed 19 DPA stage-specific increase in GUS expression. 5' deletion indicated that the 194-bp fragment between -788 and -594 relative to the transcription start site was essential for this stage-specific expression. Site-directed mutagenesis of eight transcription factor binding sites within PGhGDSL demonstrated that the MYB1AT motif (AAACCA) at -603/-598 was critical for the 19 DPA-specific reporter gene expressions. Yeast one-hybrid (Y1H) analysis identified nine proteins, including GhMYB1 (CotAD_64719) that bound to the PGhGDSL promoter. Further, Y1H experiments using the 5' promoter deletions and individually mutated promoter motifs indicated that GhMYB1 interacted with PGhGDSL at MYB1AT sequence. GhMYB1 was expressed specifically in fibre from 19 DPA, overlapping with the sharp rise in GhGDSL expression, indicating that it could regulate GhGDSL during fibre development. Analysis of genes co-expressed with GhMYB1 showed that it potentially regulates a number of other 19-25 DPA-specific genes in networks including those functioning in the cell wall and precursor synthesis, but not the major polysaccharide and protein components of the fibre SCW. GhGDSL and its promoter are therefore potential tools for the improvement of cotton fibre quality traits.

Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome.

The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide-binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large-scale identification. We present a novel proteomic peptide phage display (ProP-PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large-scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP-PD library was validated through selections against SLiM-binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM-binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non-C-terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein-protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40-130 µm through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using full-length proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PD-derived, SLiM-based PPIs. We demonstrated the methods potential to identify low affinity motif-mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery.

Studies on the interactions of SAP-1 (an N-terminal truncated form of cystatin S) with its binding partners by CD-spectroscopic and molecular docking methods.

SAP-1 is a 113 amino acid long single-chain protein which belongs to the type 2 cystatin gene family. In our previous study, we have purified SAP-1 from human seminal plasma and observed its cross-class inhibitory property. At this time, we report the interaction of SAP-1 with diverse proteases and its binding partners by CD-spectroscopic and molecular docking methods. The circular dichroism (CD) spectroscopic studies demonstrate that the conformation of SAP-1 is changed after its complexation with proteases, and the alterations in protein secondary structure are quantitatively calculated with increase of α-helices and reduction of ß-strand content. To get insight into the interactions between SAP-1 and proteases, we make an effort to model the three-dimensional structure of SAP-1 by molecular modeling and verify its stability and viability through molecular dynamics simulations and finally complexed with different proteases using ClusPro 2.0 Server. A high degree of shape complementarity is examined within the complexes, stabilized by a number of hydrogen bonds (HBs) and hydrophobic interactions. Using HB analyses in different protein complexes, we have identified a series of key residues that may be involved in the interactions between SAP-1 and proteases. These findings will assist to understand the mechanism of inhibition of SAP-1 for different proteases and provide intimation for further research.

Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: purification and enzyme kinetic properties.

The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papain-Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research.

Heparin and heparin binding proteins: potential relevance to reproductive physiology.

Glycosaminoglycans (GAGs) have crucial roles in cell-cell interaction and communication. The communication between sperm and egg during fertilization is the finest example of intercellular communication involving a protein-carbohydrate recognition system. GAGs, especially heparin, are implicated in various processes, such as capacitation, acrosome reaction (AR), and sperm nuclei decondensation by interacting with a wide range of proteins, leading to fertilization. Seminal plasma (SP) comprises of multiple proteins that bind to heparin and related GAGs. Heparin binding proteins (HBPs) originating from secretions of the male accessory sex glands are known to play a vital role during fertilization events. They interact with GAGs present in the female genital tract and enhance the subsequent zona pellucida-induced AR, and thus have been correlated with fertility in some species. Several carbohydrate and zona pellucida-binding proteins, many of which belong to the spermadhesin family, are identified as HBPs. Many studies have been documented about the potential physiological role of some HBPs in various steps of fertilization. However, there is insufficient knowledge about functions executed by various HBPs and their exact mechanism and pathways. An in-depth knowledge of HBPs and their role in fertilization is of fundamental importance to resolve biological pathways and protein interactions at the molecular level. This review surveys some of the relevant findings supporting the potential role of heparin and HBPs in reproduction. It also describes consensus heparin binding sites emerging from known literature on HBPs that interact with heparin.

Kinetic and structural studies on the interactions of heparin and proteins of human seminal plasma using surface plasmon resonance.

Heparin is naturally occurring polysaccharides which interacts with seminal plasma proteins and regulate multiple steps in fertilization process. Qualitative and quantitative information regarding the affinity for heparin-seminal plasma proteins interactions is not generally well documented and there are no reports of a comprehensive analysis of these interactions in human seminal plasma. Such information should improve our understanding of how GAGs especially heparin present in the reproductive tract regulate fertilization. In this study, we use SPR to study interactions of heparin with various seminal plasma heparin-binding proteins (HBPs). HBPs like lactoferrin (LF), fibronectin fragment (FNIII), semenogelinI (SGI) and prostate specific antigen (PSA) all bind heparin with different binding kinetics and affinities. Kinetic data suggests that FNIII binds heparin with a high affinity (KD=3.2 nM), while PSA binds heparin with a micromolar affinity (KD=11.1 µM). Preincubation of SGI with heparin inhibits the binding of SGI to immobilized PSA in a dose-dependent manner, while FNIII incubated with heparin binds with an increased affinity to PSA. Solution-competition studies show that the minimum size of a heparin oligosaccharide capable of binding with PSA is greater than a tetrasaccharide, with LF and SGI is larger than a hexasaccharide and for FNIII is larger than an octasaccharide.

Purification and characterization of a native zinc-binding high molecular weight multiprotein complex from human seminal plasma.

The seminal plasma comprises secretions from various accessory sex glands. During fertilization spermatozoa undergo complex sequences of precisely timed events that are regulated by the activation of different intracellular signaling pathways. The precision and efficacy of these pathways are often influenced by the assembly and interactions of multiprotein complexes, thereby directing the flow of regulatory information. Our knowledge about these protein complexes present in human seminal plasma (HuSP) is limited. Here we report the identification and characterization of a native high molecular weight zinc-binding multiprotein complex from HuSP by utilizing 2-DE followed by MS. Twenty-six proteins representing isoforms and/or fragments of 11 different proteins were found to be assembled in this complex. Prostate-specific antigen, zinc α2-glycoprotein, prostatic acid phosphatase, and prolactin inducible protein were the major proteins of this complex. Dynamic light scattering experiments revealed changes in aggregation pattern accompanied with deviation from physiological pH and in presence of SDS. However, no significant changes were observed in the presence of physiological ligands such as zinc and fructose. The present study will be useful and contribute to guide the future studies performed for elucidation of biological significance of this native complex in HuSP.

Differential proteomics of sperm: insights, challenges and future prospects.

Male factors account for 40% of infertility cases and most are caused by low sperm count, poor sperm quality or both. Defects in sperm are directly linked to reproductive malfunctions, and these defects may be caused by genetic mutations, environmental factors and exposure to free radicals, for example. Almost half of the male infertility cases have no known cause, indicating the lack of sensitive tests for the diagnosis of infertility. Proteomics has evolved as a major research field in biology and medicine, to identify and validate potent targets, at the molecular level, for development of more sensitive diagnostic tools. The recent advances in this field focus on the identification of differentially expressed proteins and analyzing their functional aspects for better understanding of the biological pathways. It not only provides a platform to discover biomarkers of infertility, but may also help in the design of effective male contraceptives. This article discusses various insights of proteomics for exploring biomarkers of male infertility in sperm. It also discusses the enhanced understanding of reproductive physiology offered by data produced by proteomic studies of spermatozoa.