Deep sequencing reveals divergent expression patterns within the small RNA transcriptomes of cultured and vegetative tissues of sugarcane
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ABSTRACT: We report the deep sequencing of small RNA populations derived from apex, internode 3, internode 6, internode 16, leaf +3, calli and suspesion cells from sugarcane cultivar Q117 Each vegetative tissue was sampled from three independent field grown plants, pooled and sequenced using the GAIIx platform. Calli and suspension cell cultures were sequenced using the HiSeq 2000 platform.
Project description:We report the deep sequencing of small RNA populations derived from apex, internode 3, internode 6, internode 16, leaf +3, calli and suspesion cells from sugarcane cultivar Q117
Project description:we report the identification and sequences of the tRNAome of industrially relevant microorganism Lactococcus lactis Three Next Generation sequencing runs annotated as S1, S2 and S3 were performed. Cells were harvested at exponential phase and tRNA was isolated. S1 and S2 were spiked with Phe-tRNAGAA from yeast and Lys-tRNAUUU from E. coli prior to cell lysis. S3 was spiked with Phe-tRNAGAA from yeast and Lys-tRNAUUU from E. coli before the library preparation to estimate the possible loss of tRNA in the extraction process.
Project description:Small RNAs constitute a new and unanticipated layer of gene regulation present in the three domains of life. In plants, all organs are ultimately derived from a few pluripotent stem cells localized in specialized structures called apical meristems. The development of meristems involves a coordinated balance between undifferentiated growth and differentiation, a phenomenon requiring a tight regulation of gene expression. We used in vitro cultured embryogenic calli as a model to investigate the roles of meristem-associated small RNAs. Using high-throughput sequencing, we sequenced 20 million short reads with size of 18-30nt from rice undifferentiated and differentiated calli. We confirmed 50 known microRNA families, representing one third of annotated rice microRNAs. Using a specific computational pipeline for plant microRNA identification, we identified 24 novel microRNA families. Among them, 53 microRNA or microRNA* sequences appear to vary in expression between differentiated and undifferentiated calli, suggesting a role in meristem development. Our analysis also revealed a new class of plant small RNAs derived from 5' or 3' ends of mature tRNA analogous to the tRFs in human cancer cell. We independently verified the expression of these small RNAs from 5' end of mature tRNA using qRT-PCR. Examination of 2 different small RNA expression profilings in 2 developmental stages of meristems.
Project description:The recent reports of two circular RNAs (circRNAs) with strong potential to act as microRNA (miRNA) sponges suggest that circRNAs might play important roles in regulating gene expression. However, the global properties of circRNAs are not well understood. We developed a computational pipeline to identify circRNAs and quantify their relative abundance from RNA-seq data. Applying this pipeline to a large set of non-poly(A)-selected RNA-seq data from the ENCODE project, we annotated 7,112 human circRNAs that were estimated to comprise at least 10% of the transcripts accumulating from their loci. Most circRNAs are expressed in only a few cell types and at low abundance, but they are no more cell-type–specific than are mRNAs with similar overall expression levels. Although most circRNAs overlap protein-coding sequences, ribosome profiling provides no evidence for their translation. We also annotated 635 mouse circRNAs, and although 20% of them are orthologous to human circRNAs, the sequence conservation of these circRNA orthologs is no higher than that of their flanking linear exons. The previously proposed miR-7 sponge, CDR1as, is one of only two circRNAs with more miRNA sites than expected by chance, with the next best miRNA-sponge candidate deriving from a primate-specific zinc-finger gene, ZNF91. These results provide a new framework for future investigation of this intriguing topological isoform while raising doubts regarding a biological function of most circRNAs. Examination of 9 samples in 1 cell type Note: The ENCODE data we used are under GEO SuperSeries GSE26284 (all samples labeled "_cell_total"). But they were not used in the processing of the U2OS data.
Project description:Sugarcane is an economically important crop contributing to the world’s sugar and ethanol production with 80% and 40%, respectively. In recent years, the growing demands for sugar and ethanol production has prompted the necessity to increase sugarcane productivity through conventional breeding programs. However, sugarcane breeders have encountered several difficulties to raise productivity, mainly due to its complex genetics. Sugarcane has a polyploidy genome, with many varieties being aneuploidy. Today, the majority of the planted sugarcane cultivars are complex hybrids derived mainly from crosses between Saccharum officinarum and S. spontaneum. Therefore, proteomics can provide some insight into deciphering gene regulation and changes in carbon metabolism and sucrose accumulation in the culms at different stages of plant development. The aim of this work was to compare the quantitative changes of proteins in sugarcane culms, during plant growth and sucrose accumulation. Total proteins were isolated from both, juvenile and maturing internodes at three stages of plant development. Label free shotgun proteomics was used for protein profiling and quantification. The internodes 5 (I5) and 9 (I9) of 4, 7 and 10 month-old-plants (4M, 7M and 10M, respectively) were harvested and used for proteomic analyses. To mimic field conditions of sucrose accumulation during sugarcane maturation, we stopped watering 10M plants for 10 days. An average of 1130 proteins, unique and differentially expressed across all ages were identified and quantified. Proteins were categorized within 27 functional groups, related to biological process. The patterns of expression for some categories, such as cellular amino acids, metabolic processes, secondary metabolic processes and translation were down-regulated in the immature internode (I5-10M), while up-regulated in the mature I9-10M. We observed an increase in the abundance of several enzymes of the glycolytic pathway and isoforms of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC), in the juvenile stages of development of I9. These changes in enzymes contents indicates that at the early stages of internode development, hypoxia is increasing the glycolytic and ethanolic fermentation pathways, in order to supply ATP for plant growth and NAD+ for mitochondrial respiration, which might be impaired by the low oxygen availability inside the culm.
Project description:PIWI proteins bind to PIWI-interacting RNAs (piRNAs) and play key roles in the biogenesis and functions of piRNAs. It has been reported that PIWI proteins are essential for stem cell self-renewal and germline development in diverse organisms, and are ectopically expressed in multiple forms of cancer. However, the role of PIWI in cancer remains elusive. Here we report that one of the four PIWI proteins in humans, PIWIL4, is highly expressed in both breast cancer tissues and the cytoplasm of MDA-MB-231 cell line derived from breast cancer. Reducing PIWIL4 expression drastically impairs migration ability of MDA-MB-231 cells, significantly increases their apotosis, and mildly affects their proliferation. Our transcriptome and proteome analyses reveal that these functions are at least partially achieved via the PIWIL4 regulation of TGF-beta and FGF signaling pathways and major histocompatibility complex (MHC) class II proteins. These findings suggest that PIWIL4 may serve as a potential therapeutic target for breast cancer. Examination of small RNAs in two conditions
Project description:Alcelaphine herpesvirus 1 (AlHV-1) is a ?-herpesvirus (?-HV) carried asymptomatically by wildebeest. Upon cross-species transmission, AlHV-1 induces an acute and fatal lymphoproliferative disease named malignant catarrhal fever (MCF) in many ruminants, including cattle and the rabbit model. Latency has been shown to be essential for MCF induction. However, the mechanisms causing the activation and proliferation of infected CD8+ T cells are unknown. Many ?-HVs express microRNAs (miRNAs). These small noncoding RNAs can suppress host or viral target genes involved in various pathways and are thought to facilitate viral infection and/or mediate activation and proliferation of infected lymphocytes. AlHV-1 genome has been predicted to encode a large number of miRNAs. However, their precise contribution in viral infection and pathogenesis in vivo remains unknown. Here, we have cloned small RNAs and sequenced 36 potential miRNAs expressed in a lymphoblastoid cell line propagated from a calf infected with AlHV-1 and developing MCF. Among the sequenced candidate miRNAs, 32 were expressed on the reverse strand of the genome in two main clusters. The expression of these 32 viral miRNAs was further validated using Northern blot and qRT-PCR in lymphoid organs of MCF-developing calves or rabbits. To determine the concerted contribution in MCF of 28 viral miRNAs clustered in the non-protein-coding region of the AlHV-1 genome, a recombinant virus was produced. The absence of these 28 miRNAs did not affect viral growth in vitro nor MCF induction in rabbits, demonstrating that AlHV-1 miRNAs clustered in the non-protein-coding genomic region are not essential for MCF induction. Small RNA sequencing from total RNA from AlHV-1-infected bovine lymphoblastoid cell line propagated with interleukin 2
Project description:Diverse naturally-occurring small RNA species interact with Argonaute proteins to mediate sequence-specific regulation in animals. In addition to micro-RNAs (miRNAs), which collectively regulate thousands of target mRNAs, other endogenous small RNA species include the Piwi-associated piRNAs that are important for fertility and a less well-characterized class of small RNAs often referred to simply as endo-siRNAs. Here we have utilized deep-sequencing technology and C. elegans genetics to explore the biogenesis and function of endo-siRNAs. We describe conditional alleles of the dicer-related helicase, drh-3, that implicate DRH-3 in both the response to foreign dsRNA as well as the RNA-dependent RNA Polymerase (RdRP)-dependent biogenesis of a diverse class of endogenous small RNAs, termed 22G-RNAs. We show that 22G-RNAs are abundantly expressed in the germline and maternally inherited and are the products of at least two distinct 22G-RNA systems. One system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage-related structures termed P-granules. The WAGO 22G-RNA system silences transposons, pseudogenes and cryptic loci as well as a number of genes. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one of the multiple, distinct WAGO surveillance pathways. These findings broaden our understanding of the biogenesis and diversity of 22G-RNA species and suggest potential novel regulatory functions for these small RNAs. 18 samples examined. Small RNA libraries generated from: C. elegans animals with mutations in the WAGO pathway and a WAGO-1 immunopercipitate.