Project description:Pseudouridine (Ψ) is an isomer of uridine found in ribosomal, transfer and other structural RNAs as well as in some mRNAs and non-coding RNAs, but is difficult to detect in short RNA sequences. Using modified techniques we found Ψ in microRNAs (miRNAs) and their precursors from mammalian and plant cells, primarily at the 5ʹ terminus of the mature miRNA. Small RNAs targeting transposons in reproductive cells (piRNA in testis and easiRNA in pollen) were highly enriched for Ψ, indicating a potential role in epigenetic inheritance. In pollen, pseudouridylated small RNAs were produced by RNA polymerase IV and were localized to sperm cells, as were miRNAs with terminal Ψ. We show that pseudouridylated easiRNAs from pollen contribute to imprinting and the triploid block (chromosome dosage-dependent epigenetic lethality) via the activity of PAUSED/HEN5, the plant homolog of Exportin-t. Exportin-t is required for nuclear export of pseudouridylated tRNA, and we found that PSD is required for cell-cell transport of small RNA in the germline.
Project description:Epigenetic modifications that arise during plant and animal development, such as DNA and histone modification, are mostly reset during gamete formation, but some are inherited from the germline including those marking imprinted genes. Small RNAs guide these epigenetic modifications, and some are also inherited by the next generation. In C. elegans, inherited small RNA precursors have poly (UG) tails, but how inherited small RNAs are distinguished in other animals and plants is unknown. Pseudouridine (Ψ) is the most abundant RNA modification but has not been explored in small RNAs. Here, we develop novel assays to detect Ψ in short RNA sequences, demonstrating its presence in mouse and Arabidopsis microRNAs and their precursors. We also detect substantial enrichment in germline small RNAs, namely epigenetically activated siRNAs (easiRNAs) in Arabidopsis pollen, and piwi-interacting piRNAs in mouse testis. In pollen, pseudouridylated easiRNAs are localized to sperm cells, and we found that PAUSED/HEN5 (PSD), the plant homolog of Exportin-t, interacts genetically with Ψ and is required for transport of easiRNAs into sperm cells from the vegetative nucleus. We further show that Exportin-t is required for the triploid block: chromosome dosage-dependent seed lethality that is epigenetically inherited from pollen. Thus, Ψ has a conserved role in marking inherited small RNAs in the germline.
Project description:Epigenetic modifications that arise during plant and animal development, such as DNA and histone modification, are mostly reset during gamete formation, but some are inherited from the germline including those marking imprinted genes. Small RNAs guide these epigenetic modifications, and some are also inherited by the next generation. In C. elegans, these inherited small RNAs have poly (UG) tails, but how inherited small RNAs are distinguished in other animals and plants is unknown. Pseudouridine (Ψ) is the most abundant RNA modification but has not been explored in small RNAs. Here, we developed novel assays to detect Ψ in short RNA sequences, demonstrating its presence in mouse and Arabidopsis microRNAs and their precursors. We also detected substantial enrichment in germline small RNAs, namely epigenetically activated siRNAs (easiRNAs) in Arabidopsis pollen, and PIWI-interacting piRNAs in mouse testis. In pollen, pseudouridylated easiRNAs are produced by RNA polymerase IV and localized to sperm cells. We found that PAUSED/HEN5 (PSD), the plant homolog of Exportin-t, interacts genetically with Ψ synthase and is required for transport of easiRNAs into sperm cells from the vegetative nucleus. We further show that PSD is required for the triploid block: chromosome dosage-dependent seed lethality that is epigenetically inherited from pollen. We propose that Ψ has a conserved role in marking inherited small RNAs in the germline.
Project description:To analyze the effect of Exportin-5 expression on the MEF cells in cell cycle re-entry phase, we have employed whole genome microarray expression profiling on the MEF cells in cell cycle re-entry phase with and without down regulation of Exportin-5 gene. Mouse MEF cells were transfected with 60nM of siRNA targeting either Exportin-5 or negative control, and incubated for 12 hours. After incubation, cells were starved with DMEM containing 0.2% FCS for 48 hours and re-fed with DMEM containing 20%FCS for 24 hours, along with second siRNA transfection. Two independant expreiments were preformed.
Project description:Pseudouridine is an isomer of uridine and is the most common RNA modification in both procaryotes and eucaryotes. It is found in ribosomal, transfer, and other structural RNA as well as in some mRNA and non-coding RNA. We have found abundant pseudouridine in small RNA and their precursors in Arabidopsis.
Project description:Pseudouridine is an isomer of uridine and is the most common RNA modification in both procaryotes and eucaryotes. It is found in ribosomal, transfer, and other structural RNA as well as in some mRNA and non-coding RNA. We have found abundant pseudouridine in small RNA and their precursors in Arabidopsis.
Project description:In an effort to produce a mouse model of Mitochondrial Myopathy with Lactic acidosis and Sideroblastic Anemia (MLASA), we knocked out the gene for Pseudouridine synthase 1 (PUS1), an enzyme that modifies uridine to pseudouridine in many cytoplasmic and mitochondrial tRNAs, as well as other cellular RNAs. The Pus1-/- mice are viable, are born at the expected Mendelian frequency, and are non-dysmorphic. The PUS1 mRNA and certain pseudouridine modifications are absent in cytoplasmic and mitochondrial tRNAs in the Pus1-/- mice. The Pus1-/- mice display reduce exercise capacity at 14 weeks, with alterations in muscle morphology, histology, and physiology. Red gastrocnemius muscle from Pus1-/- mice shows reduced number and size of mitochondria and reduced Cytochrome C oxidase activity. Two-condition, two-color experiment: Mouse wild type PUS1 and homozygous mutant PUS1 kidney tissue samples: 4 biological replicates each.
Project description:In an effort to produce a mouse model of Mitochondrial Myopathy with Lactic acidosis and Sideroblastic Anemia (MLASA), we knocked out the gene for Pseudouridine synthase 1 (PUS1), an enzyme that modifies uridine to pseudouridine in many cytoplasmic and mitochondrial tRNAs, as well as other cellular RNAs. The Pus1-/- mice are viable, are born at the expected Mendelian frequency, and are non-dysmorphic. The PUS1 mRNA and certain pseudouridine modifications are absent in cytoplasmic and mitochondrial tRNAs in the Pus1-/- mice. The Pus1-/- mice display reduce exercise capacity at 14 weeks, with alterations in muscle morphology, histology, and physiology. Red gastrocnemius muscle from Pus1-/- mice shows reduced number and size of mitochondria and reduced Cytochrome C oxidase activity. Two-condition, two-color experiment: Mouse wild type PUS1 and homozygous mutant PUS1 liver tissue samples: 4 biological replicates each.