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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing

ABSTRACT: To interrogate single-base resolution 6mA sites in the genome-wide, we develop DA-6mA-seq (DpnI-Assisted N6-methylAdenine sequencing), an optimized sequencing method taking advantage of restriction enzyme DpnI, which exclusively cleaves methylated adenine sites. We find DpnI also recognizes other sequence motifs besides the canonical GATC restriction sites, largely expanding the application range of this method. DA-6mA-seq requires less starting material and lower sequencing depth than previous methods, but achieves higher sensitivity, providing a good strategy to identify 6mA in large genome with a low abundance of 6mA. We rebuild the 6mA maps of Chlamydomonas by DA-6mA-seq and then apply this method to another two eukaryotic organisms, Plasmodium and Penicillium. Further analysis reveals most 6mA sites are symmetric at various sequence contexts, suggesting 6mA may function as a new heritable epigenetic mark in eukaryotes. A new sequencing method is developed to detect 6mA in eukaryotes

ORGANISM(S): Penicillium chrysogenum

SUBMITTER: Guan-Zheng Luo

PROVIDER: E-GEOD-74430 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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N6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas

Project description:N6-methyldeoxyadenosine (6mA or m6A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria, and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution, and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms. Multiple sequencing methods are developed to profile the distribution of 6mA in Chlamydomonas including MeDIP-Seq, enzyme-treated DNA-Seq, MNase-Seq and RNA-Seq.

2015-04-30 | E-GEOD-62690 | biostudies-arrayexpress

Discovery of telomere repeat-binding factor in the malaria parasite Plasmodium falciparum

Project description:ChIP-seq experiments were performed for the putative telomere repeat-binding factor (PfTRF) in the malaria parasite Plasmodium falciparum strain 3D7. The gene encoding this factor (PF3D7_1209300) was endogenously tagged with either a GFP- or a 3xHA-tag and these transgenic parasite lines were used in ChIP-sequencing experiments. Sequencing of the ChIP and input libraries showed enrichment of PfTRF at all telomere-repeat containing chromosome ends (reference genome Plasmodium falciparum 3D7 from PlasmoDB version 6.1) as well as in all upsB var promoters.In addition,PfTRF was enriched at seven additional, intra-chromosomal sites and called in the PfTRF-HA ChIP-seq only. Plasmodium falciparum 3D7 parasites were generated with -GFP or -3xHA C-terminal tagged TRF (PF3D7_1209300). Nuclei were isolated from formaldehyde cross-linked schizont-stage transgenic parasites and used to prepare chromatin. Chromatin immunoprecipitations were performed using mouse anti-GFP (Roche Diagnostics, #11814460001) or rat anti-HA 3F10 (Roche Diagnostics, #12158167001). Sequencing libraries were prepared according to a Plasmodium-optimized library preparation procedure including KAPA polymerase-mediated PCR amplification.

2016-07-15 | E-GEOD-76537 | biostudies-arrayexpress

Integrated transcriptomic and proteomic analyses of P. falciparum gametocytes: molecular insight into sex-specific processes and translational repression

Project description:Sexual differentiation of malaria parasites into gametocytes in the vertebrate host and subsequent gamete fertilisation in mosquitoes is essential for the spreading of the disease. The molecular processes orchestrating these transitions are far from fully understood. Here we report the first transcriptome analysis of male and female Plasmodium falciparum gametocytes coupled with a comprehensive proteome analysis. In male gametocytes there is an enrichment of proteins involved in the formation of flagellated gametes; proteins involved in DNA replication, chromatin organisation and axoneme formation. On the other hand, female gametocytes are enriched in proteins required for zygote formation and functions after fertilisation; protein-, lipid- and energy-metabolism. Integration of transcriptome and proteome data revealed 512 highly expressed maternal transcripts without corresponding protein expression indicating large scale translational repression in P. falciparum female gametocytes for the first time. Despite a high degree of conservation between Plasmodium species, 260 of these ârepressed transcriptsâ have not been previously described. Moreover, for some of these genes, protein expression is only reported in oocysts and sporozoites indicating that repressed transcripts can be partitioned into short- and long-term storage. Finally, these data sets provide an essential resource for identification of vaccine/drug targets and for further mechanistic studies. Examining the transcriptome of p47-mCherry positive female and pDynGFP positive (or double positive) male gametocytes by RNA-seq.

2016-06-28 | E-GEOD-75795 | biostudies-arrayexpress

Genome?wide analysis of pre?mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length: A-seq

Project description:Through alternative polyadenylation, human mRNAs acquire longer or shorter 3' untranslated regions, the latter typically associated with higher transcript stability and increased protein production. To understand the dynamics of polyadenylation site usage, we mapped transcriptome?wide both binding sites of 3' end processing factors CPSF?160, CPSF?100, CPSF?73, CPSF?30, Fip1, CstF?64, CstF-64tau, CF Im25, CF Im59, and CF Im68 and 3' end processing sites in HEK293 cells. We found that although binding sites of these factors generally cluster around the poly(A) sites most frequently used in cleavage, CstF?64/CstF-64tau and CF Im proteins have much higher positional specificity compared to CPSF components. Knockdown of CF Im68 induced a systematic use of proximal polyadenylation sites, indicating that changes in relative abundance of a single 3' end processing factor can modulate the length of 3' untranslated regions transcriptome-wide, and suggesting a mechanism behind the previously observed increase in tumor cell invasiveness upon CF Im68 knockdown. 3' ends of transcripts were profiled by high-throughput sequencing in HEK 293 cells under normal conditions, and in HEK 293 cells depleted of 3' end processing factors CF Im 68 and CstF-64.

2012-05-15 | E-GEOD-37037 | biostudies-arrayexpress

Cleavage Factor Im as a key regulator of 3’ UTR length

Project description:In eukaryotes, the 3' ends of RNA polymerase II-generated transcripts are made in the majority of cases by site-specific endonucleolytic cleavage, followed by the addition of a poly(A) tail. By alternative polyadenylation, a gene can give rise to multiple mRNA isoforms that differ in the length of their 3' UTRs and hence in their susceptibility to post-transcriptional regulatory factors such as microRNAs. A series of recently conducted high-throughput studies of poly(A) site usage revealed an extensive tissue-specific control of 3’ UTR length and drastic changes in 3’ UTR length of mRNAs upon induction of proliferation in resting cells. To understand the dynamics of polyadenylation site usage, we recently identified binding sites of the major pre-mRNA 3’ end processing factors - cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and cleavage factor Im (CF Im) - and mapped cleaved polyadenylation sites in HEK293 cells. Our present study extends previous findings on the role of CF Im in alternative polyadenylation and reveals that subunits of the CF Im complex generally control 3’ UTR length. More specifically, we demonstrate that the loss-of-function of CF Im68 and CF Im25 but not of CF Im59 leads to a transcriptome-wide increase of the use of proximal polyadenylation sites. 3' ends of transcripts were profiled by high-throughput sequencing in HEK 293 cells under normal conditions, and in HEK 293 cells depleted of 3' end processing factors CF Im25, CF Im59, and CF Im68.

2012-10-30 | E-GEOD-40137 | biostudies-arrayexpress

The systematic shortening of 3’ UTRs has limited influence on the relative change in protein abundance in proliferating cells

Project description:Alternative polyadenylation is an important cellular mechanism that enables generation of mRNA isoforms that differ in their 3' untranslated regions (3' UTRs) and consequently in their susceptibility to miRNA and RNA binding protein mediated regulation. A dramatic change in polyadenylation site usage, leading to the systematic expression of short 3’ UTR isoforms is known to occur upon induction of proliferation in resting cells. To understand the functional consequences of short 3’ UTR isoform expression we used 3' end sequencing and quantitative mass spectroscopy to determine polyadenylation site use, mRNA and protein levels in murine and human naive and activated T cells. We found that while the process and its impact on the susceptibility to miRNA and RNA binding protein mediated regulation are evolutionarily conserved, the conservation is poor at the level of individual orthologous genes. Contrary to the common belief, we did not find that transcriptome-wide 3' UTR shortening leads to a matched increase in mRNA and protein levels of genes with tandem polyadenylation sites. 3' ends of transcripts were profiled by high-throughput sequencing in murine and human naive and activated T cells.

2014-09-25 | E-GEOD-54950 | biostudies-arrayexpress

Global analyses of the effect of different cellular contexts on microRNA targeting (3P-Seq Human)

Project description:PolyA, Position, Profiling (3P-seq) for human cell lines There is no biological replicates.

2013-12-02 | E-GEOD-52527 | biostudies-arrayexpress

Global analyses of the effect of different cellular contexts on microRNA targeting (3P-Seq Mouse)

Project description:PolyA, Position, Profiling (3P-seq) for mouse tissues and cell lines There is no biological replicates. For six mouse tissues, 3P-seq libraries were constructed in wild-type and miR-22 knockout mouse.

2013-12-02 | E-GEOD-52528 | biostudies-arrayexpress

Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length

Project description:This SuperSeries is composed of the following subset Series: GSE37037: Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length: A-seq GSE37398: Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length: CLIP Refer to individual Series

2012-05-14 | E-GEOD-37401 | biostudies-arrayexpress

Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Project description:Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5' end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1-PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome. 3' end sequencing of poly (A) + RNAs in mouse ES cells with and without U1 snRNP inhibition using antisense morpholino oligonucleotides (AMO). Each with two biological replicates.

2013-06-28 | E-GEOD-46433 | biostudies-arrayexpress

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