Project description:By using dsRNA screening, we found that GW182 is critical for the degradation of circRNA in the Drosophila cells. To further confirm our finding genome widely, S2 cells were treated with either β-gal dsRNA (as a control) or GW182 dsRNA for 3 days and total RNA was extracted and sequenced after ribosome depletion. By comparing the expression level of both circRNAs and linear mRNAs between controls and GW182 depleted samples, we confirmed that GW182 depletion led to accumulation circRNAs but not their parental linear mRNAs, indicating that GW182 may play an import role in the degradation of circRNAs.
Project description:Circular RNAs (circRNAs) have emerged as crucial regulators in physiology and human diseases. However, evolutionarily conserved circRNAs with potent functions in cancers are rarely reported. Here, we identified a mammalian conserved circRNA circLARP2 that played critical roles in hepatocellular carcinoma (HCC). With clinical specimens, we found that patients with high circLARP2 levels in HCC had advanced prognostic stage and poor overall survival. CircLARP2 facilitated HCC metastasis and lipid accumulation in HCC cell lines. CircLARP2 was one of the rare ones that were identified in HCC metastasis and conserved in mammals, which enabled further studies with animal models. CircLARP2-deficient mice exhibited reduced metastasis and less lipid accumulation in an induced HCC model. We provided lines of evidence at molecular, cellular, and whole organismal levels, to support that circLARP2 functioned as a protein sponge of AUF1. CircLARP2 sequestered AUF1 from binding to LKB1 mRNA, which led to decreased LKB1 mRNA stability and lower LKB1 protein levels. LKB1 as a kinase promoted the phosphorylation of AMPK and then the phosphorylation of ACC, the rate limiting enzyme of fatty acid synthesis. Knockdown of Lkb1 with AAV8-shLkb1 in mice HCC model also proved that Lkb1 was a key element in the regulation. Through this AUF1-LKB1-AMPK-ACC pathway, circLARP2 promoted HCC metastasis and lipid accumulation.
Project description:To identify evolutionarily conserved Beta-catenin protein interactions, Beta-catenin mRNA from various metazoans was injected into Xenopus embryos and immunopurified at gastrula stage. Beta-catenin complexes were then separated on an SDS-PAGE gel and subjected mass spectrometric analysis
Project description:DNA methylation is an epigenetic modification of the vertebrate genome that contributes to transcriptional repression, imprinting, and X-chromosome inactivation. While the majority of the genome is blanketed in DNA methylation, regions known as CpG islands (CGIs) remain remarkably refractory to this modification. CpG islands are associated with roughly two thirds of gene promoters, are evolutionarily conserved, and play central roles in gene regulation, yet how they are protected from DNA methylation remains enigmatic. Based on the conserved nature of CpG islands, we have exploited genomic approaches and a transchromosomic model system to ask if DNA sequence is sufficient to specify the hypomethylated state at CpG islands when a human chromosome is newly introduced into mouse. Interestingly, this approach revealed that promoter-associated CGIs remain immensely refractory to DNA methylation regardless of the host species, in fitting with their conservation across vertebrate species and revealing that DNA sequence is a central driver in this outcome. In contrast, the methylation state of distal elements is highly variable between species and is host nucleus dependent. These alterations in methylation state at distal elements are defined by DNA nucleotide frequency and occupancy of DNA binding transcription factors, uncovering a widespread role for these features in defining the how this aspect of the epigenome forms away from gene promoters. These central principles are further supported by transplantation of mouse DNA sequences into the evolutionarily distant zebrafish genome, revealing the existence of a highly conserved and DNA encoded logic that shapes the vertebrate epigenome.
Project description:mRNA quality control mechanisms ensure fidelity of protein translation. An evolutionarily conserved component of the quality control machinery, Dom34/Pelota (Pelo), rescues stalled ribosomes. Here we show that Pelo is required for mammalian epidermal homeostasis. Our study reveals a novel role for the ribosome-rescue machinery in mammalian tissue homeostasis and an unanticipated specificity in its impact on different stem cell populations.
Project description:Extensive changes in replication timing occur during early mouse development, but their biological significance remains uncertain. To identify evolutionarily conserved features of replication timing and their relationships to epigenetic properties in humans, we profiled replication timing genome-wide in four human embryonic stem cell (hESC) lines, hESC-derived neural precursor cells (NPCs), lymphoblastoid cells, and two independently derived human induced pluripotent stem cell lines (hiPSCs). Results confirm the conservation of coordinately replicated megabase-sized units of chromosomes (replication domains) with stable cell type specific molecular boundaries that consolidate into larger replication domains during differentiation. Replication timing changes encompassed units of 400-800 kb and were coordinated with changes in transcription similar to mouse. Moreover, significant cell-type specific conservation of replication timing profiles was observed across regions of conserved synteny, despite significant species variation in the alignment of replication timing to isochore GC/LINE-1 content. Replication profiling also revealed a closer genome-wide epigenetic alignment of hESCs to mouse epiblast-derived stem cells (mEpiSCs) than to mouse ESCs. Finally, we identify a signature of chromatin modifications marking the boundaries of early replicating domains and a remarkably strong link between spatial proximity of chromatin as measured by Hi-C analysis and replication timing. Together, our results reveal evolutionarily conserved elements of the replication program in mammalian early development, demonstrate the power of replication profiling to identify important epigenetic distinctions between closely related stem cell populations (e.g. ESCs vs. EpiSCs), and strengthen the hypothesis that replication domains are structural and functional units of 3D chromosomal architecture. 8 cell types, with a total of 13 individual replicates (i.e. 5 in duplicates, 3 in single replicates)