Project description:Seeds are the basis of agriculture, yet their full transcriptional complexity has remained unknown. Here, we employ single-nucleus RNA-sequencing to characterize developing Arabidopsis thaliana seeds, with a focus on endosperm. Endosperm, the site of gene imprinting in plants, mediates the relationship between the maternal parent and embryo. We identify new cell types in the chalazal endosperm region, which interfaces with maternal tissue for nutrient unloading. We further demonstrate that the extent of parental bias of maternally expressed imprinted genes varies with cell cycle phase, and that imprinting of paternally expressed imprinted genes is strongest in chalazal endosperm. These data indicate imprinting in endosperm is heterogeneous and suggest that parental conflict, which is proposed to drive the evolution of imprinting, is fiercest at the boundary between filial and maternal tissues.
Project description:Chromatin accessibility and transcriptome profiling are the most critical tools in single-cell technologies. Here, we present Microwell-seq3, a high-throughput and easy-to-handle platform for high-sensitive single-nucleus chromatin accessibility or full-length gene expression profiling. The method combined pre-indexing and a dual-pass “chip-in-tube” strategy for nuclei loading and DNA amplification. Therefore, Microwell-seq3 does not require specialized equipment and is suitable for any molecular biology lab with simple consumables and thermal cycler. We applied Microwell-seq3 to profile the chromatin accessibility in more than 200,000 single nuclei and the full-length transcriptome in about 50,000 nuclei from multiple adult mouse tissues. Joint analysis of cell-type specific regulatory elements and total RNA uncovered cell type heterogeneity and differentially expressed genes in brain compared with previous polyadenylated transcripts capturing methods. Gene regulatory networks based on full-length transcriptome and chromatin accessibility provided improved cell-cell communications in neurons and glia. Finally, we applied Microwell-seq3 to comprehensively characterize malignant cells in spontaneous tumors from aging mouse.
Project description:Chromatin accessibility and transcriptome profiling are the most critical tools in single-cell technologies. Here, we present Microwell-seq3, a high-throughput and easy-to-handle platform for high-sensitive single-nucleus chromatin accessibility or full-length gene expression profiling. The method combined pre-indexing and a dual-pass “chip-in-tube” strategy for nuclei loading and DNA amplification. Therefore, Microwell-seq3 does not require specialized equipment and is suitable for any molecular biology lab with simple consumables and thermal cycler. We applied Microwell-seq3 to profile the chromatin accessibility in more than 200,000 single nuclei and the full-length transcriptome in about 50,000 nuclei from multiple adult mouse tissues. Joint analysis of cell-type specific regulatory elements and total RNA uncovered cell type heterogeneity and differentially expressed genes in brain compared with previous polyadenylated transcripts capturing methods. Gene regulatory networks based on full-length transcriptome and chromatin accessibility provided improved cell-cell communications in neurons and glia. Finally, we applied Microwell-seq3 to comprehensively characterize malignant cells in spontaneous tumors from aging mouse.
Project description:During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA (gRNA). While the simple retrovirus MLV segregates its full-length RNA into two functional populations, the HIV-1 full-length RNA was proposed to exist as a single population used indistinctly for protein synthesis or packaging. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that an epitranscriptomic switch involving the demethylation of adenosine residues present within HIV-1 5´-UTR regulates full-length RNA packaging. We further identified two conserved adenosines within the 5’-UTR that have a crucial structural and functional role and that are modulated by N6-methylation. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promotes the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus contributing to full-length RNA demethylation. Finally, the specific inhibition of the FTO RNA demethylase activity suppressed HIV-1 full-length RNA packaging. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the full-length RNA molecules that will be used as viral genomes.
Project description:To analyse the regulation of transcripts in grain embryo and endosperm during development, we performed RNA-Seq for wheat from 14 and 25 day post anthesis (DPA). And long-read sequencing for mixed whole grains from 14 and 25 DPA was employed to obtain full-length transcripts. A series of differentially expressed genes and tissues-specific genes of embryo and endosperm were identified. Moreover, 4351, 4641, 4516 and 4453 genes with A, B and D homoeoloci were detected in the four tissues. These provide specific gene pools of embryo and endosperm and homoeolog expression bias model in a large scale, which provides new insights into the molecular physiology of wheat.
Project description:Full-Length cDNA transcriptome (Iso-Seq) data sequenced on the PacBio Sequel system using 2.1 chemistry. Multiplexed cDNA library of 12 samples (3 tissues x 4 strains). Tissues: root, embryo, endosperm. Strains: B73, Ki11, B73xKi11, Ki11xB73.
Project description:The long non-coding RNA Malat1 has been implicated in several human cancers, while the mechanism of action is not completely understood. As RNAs in cells function in the context of RBPs identification of their RNA-binding proteins can shed light on their functionality. We here performed quantitative interactomics of 14 non-overlapping fragments covering the full length of Malat1 to identify possible nuclear interacting proteins. Overall, we identified 35 candidates including 14 already known binders, which are able to interact with Malat1 in the nucleus. Furthermore, the use of fragments along the full-length RNA allowed us to reveal two hotspots for protein binding, one in the 5’-region and one in the 3’-region of Malat1. Our results provide confirmation on previous RNA-protein interaction studies and suggest new candidates for functional investigations.