Project description:Emerging and neglected pathogens pose challenges as their biology is frequently poorly understood, and genetic tools often do not exist to manipulate them. Organism agnostic sequencing technologies offer a promising approach to understand the molecular processes underlying these diseases. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), the obligate intracellular causative agent of the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning, we investigated the transcriptional architecture of Ot, including operon structure and non-coding RNAs, and found evidence for wide-spread post-transcriptional antisense regulation. We compared the host response to two clinical isolates and identified distinct immune response networks that are up-regulated in response to each strain, leading to predictions of relative virulence which were confirmed in a mouse infection model. Thus, dual RNA-seq can reveal the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.
Project description:Recent studies have revealed the importance of long noncoding RNAs (lncRNAs) as tissue-specific regulators of gene expression. There is ample evidence that distinct types of vasculature undergo tight transcriptional control to preserve their structure, identity, and functions. We determined, for the first time, the global lineage-specific lncRNAome of human dermal blood and lymphatic endothelial cells (BECs and LECs), combining RNA-Seq and CAGE-Seq. A subsequent genome-wide antisense oligonucleotide-knockdown profiling of two BEC- and two LEC-specific lncRNAs identified LETR1 as a critical gatekeeper of the global LEC transcriptome. Deep RNA-DNA and RNA-protein interaction studies, and phenotype rescue analyses revealed that LETR1 is a nuclear trans-acting lncRNA modulating, via key epigenetic factors, the expression of essential target genes governing the growth and migratory ability of LECs. Together, our study provides new evidence supporting the intriguing concept that every cell type expresses precise lncRNA signatures to control lineage-specific regulatory programs.
Project description:mTOR activation has been known to affect protein synthesis. To identify molecular signatures in transcriptome that could enhance protein synthesis, RNA-seq and quantitative proteomics studies were conducted using WT and TSC1 null MEFs. In this study, we found that the activation of mTOR leads to genome-wide 3'UTR shortening in mRNAs by alternative polyadenylation and activates ubiquitin-mediated proteolysis. The accession number for the RNA-seq data in this study is SRP056624.