Unique small RNA signatures uncovered in the tammar wallaby genome [small RNA_seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Macropus eugenii
SUBMITTER: James Lindsay
PROVIDER: E-GEOD-30370 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Small RNA isolation of miRNAs, crasiRNAs and piRNAs for RNAseq from tammar wallaby pouch young
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Project description:In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (Pol) Pol IV and Pol V. Pol IV plays a major role in siRNA biogenesis, while Pol V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions which are dependent on both Pol IV and Pol V have been identified previously, the genomic loci targeted Pol V for siRNA accumulation and silencing have not been described extensively. To characterize the Pol V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and Pol V mutant plants. Furthermore, we characterized the siRNA-generating regions which were dependent on RdDM effectors and examined their dependency on Pol V. Small RNA libraries were generated and deeply sequenced from mutant alleles dms4-1, drd1-1, dms3-1, and rdm1-4, along with their control library (“Wt(T+S)”) which has been described previously (Kanno et al. 2004 Current Biology). More than 2,000 small RNA-generating loci were identified which were greatly suppressed in Pol V mutants. The Pol V-dependent, heterochromatic siRNA-generating regions were characterized in the Arabidopsis genome by deep sequencing the small RNA populations of wild-type and Pol V mutant plants. Deep SBS sequencing was used for small RNA profiling of immature inflorescence tissues from RNA polymerase V and RdDM mutants.
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Project description:A silencing signal in plants with an RNA specificity determinant moves through plasmodesmata and the phloem. To identify the mobile RNA we grafted Arabidopsis thaliana shoots to roots that would be a recipient for the silencing signal. Using high throughput sequencing as a sensitive detection method and mutants to block small RNA (sRNA) biogenesis in either source or recipient tissue, we detected endogenous and transgene specific sRNA that moved across the graft union. Surprisingly we found that the mobile endogenous sRNAs account for a substantial proportion of the sRNA in roots and we provide evidence that 24nt mobile sRNAs direct epigenetic modifications in the genome of the recipient cells. Mobile sRNA thus represents a mechanism for transmitting the specification of epigenetic modification and could affect genome defence and responses to external stimuli that have persistent effects in plants. Keywords: Small RNA Analysis, Epigenetics 34 unique samples, 15 Biological Replicates
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Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Petunia x hybrida tissues (including leaves and flowers). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the genome under study. Small RNA libraries were derived from leaves and flowers (see Series GSE13764) of Petunia x hybrida. Total RNA was isolated using the Plant RNA Purification Reagent (Invitrogen), and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Ana Dorantes-Acosta and Richard A. Jorgensen for providing the plant material as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.
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Project description:Pseudomonas aeruginosa is a highly adaptable bacterium which thrives in a broad range of ecological niches and can infect multiple hosts as diverse as plants, nematodes and mammals. In humans, it is an important opportunistic pathogen. This wide adaptability correlates with its broad genetic diversity. In this study, we used a deep-sequencing approach to explore the complement of small RNAs (sRNAs) in P. aeruginosa as the number of such regulatory molecules previously identified in this organism is relatively low, considering its genome size, phenotypic diversity and adaptability. We have performed a comparative analysis of PAO1 and PA14 strains which share the same host range but differ in pathogenicity, PA14 being considerably more virulent in several model organisms. Altogether, we have identified more than 150 novel candidate sRNAs and validated a third of them by Northern blotting. Interestingly, a number of these novel sRNAs are strain-specific or showed strain-specific expression, strongly suggesting that they could be involved in determining specific phenotypic traits. 2 cDNA samples (enriched for different size ranges ) from each of two strains of Pseudomonas aeruginoasa (PAO1 and PA14) were sequenced with the Roche 454 technology
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Project description:microRNAs (miRNAs) are a large class of small non-coding RNAs which post-transcriptionally regulate the expression of a large fraction of all animal genes and are important in a wide range of biological processes. Recent advances in high-throughput sequencing allow miRNA detection at unprecedented sensitivity, but the computational task of accurately identifying the miRNAs in the background of sequenced RNAs remains challenging. For this purpose we have designed miRDeep2, a substantially improved algorithm which identifies canonical and non-canonical miRNAs such as those derived from transposable elements and informs on high-confidence candidates that are detected in multiple independent samples. Analyzing data from seven animal species representing the major animal clades, miRDeep2 identified miRNAs with an accuracy of 98.6-99.9% and reported hundreds of novel miRNAs. To test the accuracy of miRDeep2, we knocked down the miRNA biogenesis pathway in a human cell line and sequenced small RNAs before and after. The vast majority of the >100 novel miRNAs expressed in this cell line were indeed specifically down-regulated, validating most miRDeep2 predictions. Last, a new miRNA expression profiling routine, low time and memory usage and user-friendly interactive graphic output can make miRDeep2 useful to a wide range of researchers." high-throughput sequencing was used to profile small RNA expression in a human MCF-7 cell line before and after Dicer knock-down
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Project description: Not available
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Project description:Small non-coding RNAs (sncRNAs) have been proposed as potential vectors of the interface between genes and environment. Here, we report that environmental conditions involving traumatic stress in early life, alter miRNA and piRNA composition in sperm of adult males in mice. Examination of small RNA content of sperm from males, that experienced early chronic stress during their first two weeks of life versus small RNA content of sperm from control males.
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Project description:The most experiments about buccal pouch mucosal cells were investigated in mice and hamster. In these studies we have used porcine buccal pouch mucosal cells cultured primarily in vitro to investigate the expression profile of new molecular markers of mucosal wounding, vascularization and proliferation. In this study, we demonstrated the gene expression profile of long time primary cultured porcine buccal pouch mucosal cells.
2018-04-05 | GSE112659 | GEO
Project description:Post-transcriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a method based on RNA-protein crosslinking, to identify transcriptome wide ~26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in splicing. Upon HuR knock down, mRNA levels and protein synthesis of thousands of target genes was down regulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knock down triggered strong and specific up regulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing. PolyA mRNA was extracted from anti HuR siRNA treated and mock transfected HeLa cells to identify changes in mRNA expression and splicing. 2x100 paired end sequencing was performed according to the protocol on the Illumina HiSeq. PARCLIP was performed as in Hafner et. Al 2010 but with an antibody against endogenous HuR (3A2, Santa Cruz, sc-5261) in unstressed HeLa cells. We used, independently, 4-thiouridine (4SU) and 6-thioguanosine (6SG) to assess a possible nucleotide bias. As our proteomics measurements required labeling of cells in a special medium we also performed PAR-CLIP on cells grown in SILAC medium. Altogether we performed three PAR-CLIP experiments: 4SU labeling in standard DMEM medium, 4SU labeling in SILAC medium (as a replicate) and 6SG labeling in SILAC medium. Small RNA was extracted from anti HuR siRNA treated and mock transfected HeLa cells to identify changes in mRNA expression. Sequencing was performed on Illumina GAII using the standard sRNA 36cycle protocol.
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