Similar Datasets
Project description:For the bilateral intrastriatal delivery of AAV expressing control or TRAX shRNA, mice were anesthetized with ketamine/xylazine HCl (87.56 and 7.5 ug/kg, respectively, via an intraperitoneal injection) and injected with the indicated AAV (2 µl per injection spot, 2.5*10^9 vector genome/µl) with a 10 ml syringe (Hamilton, Reno, NV, USA) at a rate of 0.5 ul/min. The AAV was injected at the desired position: AP+0.5, L±2, DV-2.5 and -3.5 mm relative to bregma and the dura surface. Then the mouse brains were removed from the mice directly for dissection of striatum. RNA was extracted by TRIzol reagent or miRNeasy mini kit (Qiagen). DNase digestion was performed by the DNA-free™ Kit (Life Technologies, Carlsbad, CA, USA) or the RNase-free DNase set (Qiagen) following the manufacturer’s instructions. The concentration and purity of RNA were measured at 260, 280 and 230 nm by a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). Sample libraries were prepared using the QIAseq miRNA Library Kit (QIAGEN) according to the manufacturer's protocols. Adaptors are ligated sequentially to the 3' and 5' ends of miRNAs. Subsequently, universal cDNA synthesis with UMI assignment, cDNA cleanup, library amplification and library cleanup are performed. Libraries were sequenced on an Illumina instrument (75-cycle single-end read, 75SE). Sequencing data was processed using the Illumina software BCL2FASTQ v2.20. Initially, the sequences generated went through a filtering process to obtain qualified reads. Trimmomatic (1) was implemented to clip the 3′ adaptor sequence, trim or remove the reads according to the quality score and discard trimmed reads shorter than 18 nucleotides. Qualified reads after filtering low-quality data were analyzed using miRDeep2 (2) for aligning reads to the reference genome downloaded from UCSC. Only reads that mapped perfectly to the genome five or less times were used for miRNA detection, since miRNAs usually map to few genomic locations. MiRDeep2 estimates expression levels of known miRNAs, and also identifies novel miRNAs. The small RNA library construction, NGS deep sequencing and bioinformatic analysis were accomplished by Welgene Biotech. Co., Ltd. (Taipei, ROC).
2024-10-14 | GSE187609 | GEO
Project description:For the bilateral intrastriatal delivery of AAV expressing control or TRAX shRNA, mice were anesthetized with ketamine/xylazine HCl (87.56 and 7.5 ug/kg, respectively, via an intraperitoneal injection) and injected with the indicated AAV (2 µl per injection spot, 2.5*10^9 vector genome/µl) with a 10 ml syringe (Hamilton, Reno, NV, USA) at a rate of 0.5 ul/min. The AAV was injected at the desired position: AP+0.5, L±2, DV-2.5 and -3.5 mm relative to bregma and the dura surface. Then the mouse brains were removed from the mice directly for dissection of striatum. RNA was extracted by TRIzol reagent or miRNeasy mini kit (Qiagen). DNase digestion was performed by the DNA-free™ Kit (Life Technologies, Carlsbad, CA, USA) or the RNase-free DNase set (Qiagen) following the manufacturer’s instructions. The concentration and purity of RNA were measured at 260, 280 and 230 nm by a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). RNA was qualified by a Bioanalyzer 2100 (Agilent Technology, Santa Clara, CA, USA) with an RNA 6000 LabChip kit (Agilent Technology). For mRNA, the RNA was prepared based on Illumina’s official protocol. Library construction was carried out by TruSeq Stranded Total RNA Library Prep Gold Kit (Illumina, San Diego, CA, USA) followed by AMPure XP beads (Beckman Coulter, Brea, CA, USA) size selection. Libraries were sequenced using sequencing-by-synthesis (SBS) technology (Illumina). Sequencing data and FASTQ reads were generated using an inhouse Welgene ’Biotech pipeline according to Illumina’s base calling program bcl2fastq v2.20.
2024-10-14 | GSE187608 | GEO
Project description:The miRNA/mRNA regulation by Translin associated factor X (TRAX) in WT and R6/2 HD mice
| PRJNA777614 | ENA
Project description:miRNA-seq for WT and R6/2 (HD) mice with Translin-associated protein X (TRAX) knockdown
| PRJNA777623 | ENA
Project description:Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity.
| S-EPMC3299731 | biostudies-literature
Project description:Translin and Trax proteins are highly conserved nucleic acid binding proteins that have been implicated in RNA regulation in a range of biological processes including tRNA processing, RNA interference, microRNA degradation during oncogenesis, spermatogenesis and neuronal regulation. Here, we explore the function of this paralogue pair of proteins in the fission yeast. Using transcript analysis we demonstrate a reciprocal mechanism for control of telomere-associated transcripts. Mutation of tfx1+ (Trax) elevates transcript levels from silenced sub-telomeric regions of the genome, but not other silenced regions, such as the peri-centromeric heterochromatin. In the case of some sub-telomeric transcripts, but not all, this elevation is dependent on the Trax paralogue, Tsn1 (Translin). In a reciprocal fashion, Tsn1 (Translin) serves to repress levels of transcripts (TERRAs) from the telomeric repeats, whereas Tfx1 serves to maintain these elevated levels. This reveals a novel mechanism for the regulation of telomeric transcripts. We extend this to demonstrate that human Translin and Trax also control telomere-associated transcript levels in human cells in a telomere-specific fashion.
| S-EPMC5085120 | biostudies-literature
Project description:RNA-seq for WT and R6/2 (HD) mice with Translin-associated protein X (TRAX) knockdown
| PRJNA777622 | ENA
Project description:The vertebrate RNA and ssDNA-binding protein Translin has been suggested to function in a variety of cellular processes, including DNA damage response, RNA transport, and translational control. The Translin-associated factor X (Trax) interacts with Translin, and Trax protein stability depends on the presence of Translin. To determine the function of the Drosophila Translin and Trax, we generated a translin null mutant and isolated a trax nonsense mutation. translin and trax single and double mutants are viable, fertile, and phenotypically normal. Meiotic recombination rates and chromosome segregation are also not affected in translin and trax mutants. In addition, we found no evidence for an increased sensitivity for DNA double-strand damage in embryos and developing larvae. Together with the lack of evidence for their involvement in DNA double-strand break checkpoints, this argues against a critical role for Translin and Trax in sensing or repairing such DNA damage. However, Drosophila translin is essential for stabilizing the Translin interaction partner Trax, a function that is surprisingly conserved throughout evolution. Conversely, trax is not essential for Translin stability as trax mutants exhibit normal levels of Translin protein.
| S-EPMC1667053 | biostudies-literature
Project description:Translin is a human octameric protein that specifically binds the single-stranded microsatellite repeats d(GT)n and the corresponding transcripts (GU)n. It also binds, with lesser affinities, other single-stranded G-rich DNA and RNA sequences. TRAX is a human protein that bears a homology to Translin and interacts with it. Translin and TRAX have been proposed to be involved in DNA recombination, chromosomal translocation and mRNA transport and translation. Both proteins are highly conserved in eukaryotes, including the fission yeast Schizosaccharomyces pombe, which is amenable to genetic analysis. Here, we report the first study of the S.pombe Translin and TRAX homologs. We have deleted the genes encoding Translin and TRAX in S.pombe and found that the proliferation of the mutant cells was slightly stimulated, suggesting that these genes are not essential for the fission yeast. We have also shown that the S.pombe Translin and TRAX interact. Biochemical analysis of the S.pombe Translin, which was cloned and expressed in Escherichia coli, revealed that it is octameric and that it selectively binds d(GT)n and d(GTT)n microsatellite repeats. However, unlike the human protein, it has much higher affinities for the homologous RNA sequences (GU)n and (GUU)n. These data suggest that the S.pombe Translin is primarily involved in functions related to RNA metabolism.
| S-EPMC1180670 | biostudies-literature
Project description:Conserved translin-TRAX complexes, also known as C3POs, have been implicated in many biological processes, but how they function remains unclear. Recently, C3PO was shown to be an endoRNase that promotes RNA interference (RNAi) in animal cells. Here, we show that C3PO does not play a significant role in RNAi in the filamentous fungus Neurospora crassa. Instead, the Neurospora C3PO functions as an RNase that removes the 5' pre-tRNA fragments after the processing of pre-tRNAs by RNase P. In addition, translin and trax mutants have elevated levels of tRNA and protein translation and are more resistant to a cell death-inducing agent. Finally, we show that C3PO is also involved in tRNA processing in mouse embryonic fibroblast cells. This study identifies the endogenous RNA substrates of C3PO and provides a potential explanation for its roles in apparently diverse biological processes.
| S-EPMC3414638 | biostudies-literature