Poly-Adenylation Lengthening of Short RNAs for Simultaneous Short and Long Nanopore Sequencing (PALS-NS)
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA928732 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23254862_1.fastq.gz | Fastqsanger.gz | |||
| SRR23254863_1.fastq.gz | Fastqsanger.gz | |||
| SRR23254864_1.fastq.gz | Fastqsanger.gz | |||
| SRR23254865_1.fastq.gz | Fastqsanger.gz | |||
| SRR23254866_1.fastq.gz | Fastqsanger.gz |
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Project description:Sequencing of long coding RNAs informs about the abundance and the novelty in the transcriptome, while sequencing of short coding RNAs (e.g., microRNAs) or long non-coding RNAs informs about the epigenetic regulation of the transcriptome. Currently, each of these goals is addressed by separate sequencing experiments given the different physical characteristics of RNA species from biological samples. Sequencing of both short and long RNAs from the same experimental run has not been reported for long-read Nanopore sequencing to date and only recently has been achieved for short-read (Illumina) methods. We propose a library preparation method capable of simultaneously profiling short and long RNA reads in the same library on the Nanopore platform and provide the relevant bioinformatics workflows to support the goals of RNA quantification. Using a variety of synthetic samples we demonstrate that the proposed method can simultaneously detect short and long RNAs in a manner that is linear over 5 orders of magnitude for RNA abundance and three orders of magnitude for RNA length. In biological samples the proposed method is capable of profiling a wider variety of short and long non-coding RNAs when compared against the existing Smart-seq protocols for Illumina and Nanopore sequencing.
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Project description:We used RNA-seq to identify gene expression changes in C. elegans pals-22 mutants, pals-22 pals-25 double mutants, and pals-25 mutants
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Project description:The immune system continually battles against pathogen-induced pressures, which often leads to the evolutionary expansion of immune gene families in a species-specific manner. For example, the pals gene family expanded to 39 members in the Caenorhabditis elegans genome, in comparison to a single mammalian pals ortholog. Our previous studies have revealed that two members of this family, pals-22 and pals-25, act as antagonistic paralogs to control the Intracellular Pathogen Response (IPR). The IPR is a protective transcriptional response, which is activated upon infection by two molecularly distinct natural intracellular pathogens of C. elegans – the Orsay virus and the fungus Nematocida parisii from the microsporidia phylum. In this study, we identify a previously uncharacterized member of the pals family, pals-17, as a newly described negative regulator of the IPR. pals-17 mutants show constitutive upregulation of IPR gene expression, increased immunity against intracellular pathogens, as well as impaired development and reproduction. We also find that two other previously uncharacterized pals genes, pals-20 and pals-16, are positive regulators of the IPR, acting downstream of pals-17. These positive regulators reverse the effects caused by the loss of pals-17 on IPR gene expression, immunity and development. We show that the negative IPR regulator protein PALS-17 and the positive IPR regulator protein PALS-20 colocalize inside intestinal epithelial cells, which are the sites of infection for IPR-inducing pathogens. In summary, our study demonstrates that several pals genes from the expanded pals gene family act as ON/OFF switch modules to regulate a balance between organismal development and immunity against natural intracellular pathogens in C. elegans.
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Project description:The immune system continually battles against pathogen-induced pressures, which often leads to the evolutionary expansion of immune gene families in a species-specific manner. For example, the pals gene family expanded to 39 members in the Caenorhabditis elegans genome, in comparison to a single mammalian pals ortholog. Our previous studies have revealed that two members of this family, pals-22 and pals-25, act as antagonistic paralogs to control the Intracellular Pathogen Response (IPR). The IPR is a protective transcriptional response, which is activated upon infection by two molecularly distinct natural intracellular pathogens of C. elegans – the Orsay virus and the fungus Nematocida parisii from the microsporidia phylum. In this study, we identify a previously uncharacterized member of the pals family, pals-17, as a newly described negative regulator of the IPR. pals-17 mutants show constitutive upregulation of IPR gene expression, increased immunity against intracellular pathogens, as well as impaired development and reproduction. We also find that two other previously uncharacterized pals genes, pals-20 and pals-16, are positive regulators of the IPR, acting downstream of pals-17. These positive regulators reverse the effects caused by the loss of pals-17 on IPR gene expression, immunity and development. We show that the negative IPR regulator protein PALS-17 and the positive IPR regulator protein PALS-20 colocalize inside intestinal epithelial cells, which are the sites of infection for IPR-inducing pathogens. In summary, our study demonstrates that several pals genes from the expanded pals gene family act as ON/OFF switch modules to regulate a balance between organismal development and immunity against natural intracellular pathogens in C. elegans.
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Project description:Early development depends heavily on accurate control of maternally inherited mRNAs, and yet it remains unknown how maternal microRNAs (miRNAs) are regulated during maternal to zygotic transition (MZT). We here find that maternal miRNAs are highly adenylated at their 3' ends in mature oocytes and early embryos. Pervasive adenylation is observed in oocytes of fly, sea urchin and mouse, indicating that maternal miRNA adenylation may be widely conserved in animals. We identify Wispy as the enzyme responsible for miRNA adenylation in flies. Wispy is known to be expressed specifically in oocytes and early embryos and function as a noncanonical poly(A) polymerase. Knockout of wispy abrogates miRNA adenylation and induces miRNA accumulation in fly eggs whereas overexpression of Wispy increases adenylation and reduces miRNA levels in S2 cells. Adenylation occurs on both the 5p and 3p miRNAs, indicating that Wispy acts on miRNAs after Dicer processing. We further find that Wispy interacts with Ago1 through protein-protein interaction, which may allow the effective and selective adenylation of miRNAs. Thus, adenylation may contribute to the clearance of maternally deposited miRNAs during MZT. Our work provides the first mechanistic insights into the regulation of maternal miRNAs and illustrates the importance of RNA tailing in development. MiRNA expression and modification profile during early embryo development of fruit fly and zebra fish using high throughput sequencing
2014-10-01 | E-GEOD-61931 | biostudies-arrayexpress
Project description:Early development depends heavily on accurate control of maternally inherited mRNAs, and yet it remains unknown how maternal microRNAs (miRNAs) are regulated during maternal to zygotic transition (MZT). We here find that maternal miRNAs are highly adenylated at their 3' ends in mature oocytes and early embryos. Pervasive adenylation is observed in oocytes of fly, sea urchin and mouse, indicating that maternal miRNA adenylation may be widely conserved in animals. We identify Wispy as the enzyme responsible for miRNA adenylation in flies. Wispy is known to be expressed specifically in oocytes and early embryos and function as a noncanonical poly(A) polymerase. Knockout of wispy abrogates miRNA adenylation and induces miRNA accumulation in fly eggs whereas overexpression of Wispy increases adenylation and reduces miRNA levels in S2 cells. Adenylation occurs on both the 5p and 3p miRNAs, indicating that Wispy acts on miRNAs after Dicer processing. We further find that Wispy interacts with Ago1 through protein-protein interaction, which may allow the effective and selective adenylation of miRNAs. Thus, adenylation may contribute to the clearance of maternally deposited miRNAs during MZT. Our work provides the first mechanistic insights into the regulation of maternal miRNAs and illustrates the importance of RNA tailing in development.
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Project description:We used RNA-seq to identify gene expression changes in C. elegans pals-25(icb98gf) mutants
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Project description:This experimental series concerns the position bias and extent of 3'-end choice in MCF7 cells after knock-down of the CFII subunits PCF11 and CLP1 in MCF7. Loss of either subunit results in 3'-lengthening by APA. Importantly this lengthening is toward a more 'normal-like' distribution. MCF7 cells show a native 3'-end distribution favoring short internal adenylation sites compared to MDAMD231 cells. The difference between the two cell lines might be driven by a higher expression of the exosome in MDAMD231 cells.
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