Biology subjects

Erle, D. J.

Publications and source records attributed to Erle, D. J..

4 recordsLinked to original sources

bioRxiv2018

A global map of RNA binding protein occupancy guides functional dissection of post-transcriptional regulation of the T cell transcriptome

RNA binding proteins (RBPs) mediate constitutive RNA metabolism and gene specific regulatory interactions. To identify RNA cis-regulatory elements, we developed GCLiPP, a biochemical technique for detecting RBP occupancy transcriptome-wide. GCLiPP sequence tags corresponded with known RBP binding sites, specifically correlating to abundant cytosolic RBPs. To demonstrate the utility of our occupancy profiles, we performed functional dissection of 3' UTRs with CRISPR/Cas9 genome editing. Two RBP occupied sites in the CD69 3' UTR destabilized the transcript of this key regulator of lymphocyte tissue egress. Comparing human Jurkat T cells and mouse primary T cells uncovered hundreds of biochemically shared peaks of GCLiPP signal across homologous regions of human and mouse 3' UTRs, including a cis-regulatory element that governs the stability of the mRNA that encodes the proto-oncogene PIM3 in both species. Our GCLiPP datasets provide a rich resource for investigation of post-transcriptional regulation in the immune system.

genomics↗

bioRxiv2018

Detection of succinate by intestinal tuft cells triggers a type 2 innate immune circuit

Initiation of immune responses requires innate immune sensing, but immune detection of the helminths, protists, and allergens that stimulate type 2 immunity remains poorly understood. In the small intestine, type 2 immune responses are regulated by a tuft cell-ILC2 signaling circuit. Tuft cells express components of a canonical taste transduction pathway, including the membrane channel TRPM5, but the ligands and receptors that activate tuft cells in the small intestine are unknown. Here we identify succinate as the first ligand that activates intestinal tuft cells to initiate type 2 immune responses. Using mRNA-Seq on tuft cells from different tissues, we show that all tuft cells express the intracellular taste transduction pathway, but expression of upstream receptors is tissue-specific. In the small intestine, tuft cells express the succinate receptor SUCNR1. Remarkably, providing succinate in drinking water is sufficient to induce a multifaceted type 2 immune response in the murine small intestine, involving all known components of the tuft-ILC2 circuit. The helminth Nippostrongylus brasiliensis secretes succinate as a metabolite, and sensing of both succinate and N. brasiliensis requires tuft cells and TRPM5, suggesting a novel paradigm in which type 2 immunity monitors microbial metabolism. Manipulation of succinate sensing may have therapeutic benefit in numerous intestinal diseases.

immunology↗

bioRxiv2018

Large differences in small RNA composition between human biofluids

Extracellular miRNAs and other small RNAs are implicated in cellular communication and may be useful as disease biomarkers. We systematically compared small RNAs in 12 human biofluid types using RNA-seq. miRNAs and tRNA-derived RNAs (tDRs) accounted for the majority of mapped reads in all biofluids, but the ratio of miRNA to tDR reads varied from 72 in plasma to 0.004 in bile. miRNA levels were highly correlated across all biofluids but levels of some miRNAs differed markedly between biofluids. tDR populations differed extensively between biofluids. Y RNA fragments were seen in all biofluids and accounted for >10% of reads in blood plasma, serum, and CSF. Reads mapping exclusively to piRNAs were very rare except in seminal plasma. These results demonstrate extensive differences in small RNAs between human biofluids and provide a useful resource for investigating extracellular RNA biology and developing biomarkers.

genomics↗

bioRxiv2017

Accuracy, Reproducibility And Bias Of Next Generation Sequencing For Quantitative Small RNA Profiling: A Multiple Protocol Study Across Multiple Laboratories

Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols.

genomics↗