Biology subjects

Zamore, P. D.

Publications and source records attributed to Zamore, P. D..

6 recordsLinked to original sources

bioRxiv2018

The RNA-binding ATPase, Armitage, Couples piRNA Amplification in Nuage to Phased piRNA Production on Mitochondria

PlWI-interacting RNAs (piRNAs) silence transposons in Drosophila ovaries, ensuring female fertility. Two coupled pathways generate germline piRNAs: the ping-pong cycle, in which the PIWI proteins Aubergine and Ago3 increase the abundance of pre-existing piRNAs, and the phased piRNA pathway, which generates strings of tail-to-head piRNAs, one after another. Proteins acting in the ping-pong cycle localize to nuage, whereas phased piRNA production requires Zucchini, an endonuclease on the mitochondrial surface. Here, we report that Armitage (Armi), an RNA-binding ATPase localized to both nuage and mitochondria, links the ping-pong cycle to the phased piRNA pathway. Mutations that block phased piRNA production deplete Armi from nuage. Armi ATPase mutants cannot support phased piRNA production and inappropriately bind mRNA instead of piRNA precursors. We propose that Armi shuttles between nuage and mitochondria, feeding precursor piRNAs generated by Ago3 cleavage into the Zucchini-dependent production of Aubergine- and Piwi-bound piRNAs on the mitochondrial surface.

genetics↗

bioRxiv2018

An Evolutionarily Conserved piRNA-producing Locus Required for Male Mouse Fertility

Pachytene piRNAs, which comprise >80% of small RNAs in the adult mouse testis, have been proposed to bind and regulate target RNAs like miRNAs, cleave targets like siRNAs, or lack biological function altogether. Although piRNA pathway protein mutants are male sterile, no biological function has been identified for any mammalian piRNA-producing locus. Here, we report that males lacking piRNAs from a conserved mouse pachytene piRNA locus on chromosome 6 (pi6) produce sperm with defects in capacitation and egg fertilization. Moreover, heterozygous embryos sired by pi6-/- fathers show reduced viability in utero. Molecular analyses suggest that pi6 piRNAs repress gene expression by cleaving mRNAs encoding proteins required for sperm function. pi6 also participates in a network of piRNA-piRNA precursor interactions that initiate piRNA production from a second piRNA locus on chromosome 10 as well as pi6 itself. Our data establish a direct role for pachytene piRNAs in spermiogenesis and embryo viability.\n\nHighlightsO_LINormal male mouse fertility and spermiogenesis require piRNAs from the pi6 locus\nC_LIO_LISperm capacitation and binding to the zona pellucida of the egg require pi6 piRNAs\nC_LIO_LIHeterozygous embryos sired by pi6-/- fathers show reduced viability in utero\nC_LIO_LIDefects in pi6 mutant sperm reflect changes in the abundance of specific mRNAs.\nC_LI

genetics↗

bioRxiv2018

An Automated Bayesian Pipeline for Rapid Analysis of Single-Molecule Binding Data

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. We benchmarked the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also used the pipeline to extend our understanding of TtAgo by measuring the proteins binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites.

molecular biology↗

bioRxiv2018

Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers

RNA-seq and small RNA-seq are powerful, quantitative tools to study gene regulation and function. Common high-throughput sequencing methods rely on polymerase chain reaction (PCR) to expand the starting material, but not every molecule amplifies equally, causing some to be overrepresented. Unique molecular identifiers (UMIs) can be used to distinguish undesirable PCR duplicates derived from a single molecule and identical but biologically meaningful reads from different molecules. We have incorporated UMIs into RNA-seq and small RNA-seq protocols and developed tools to analyze the resulting data. Our UMIs contain stretches of random nucleotides whose lengths sufficiently capture diverse molecule species in both RNA-seq and small RNA-seq libraries generated from mouse testis. Our approach yields high-quality data while allowing unique tagging of all molecules in high-depth libraries. Using simulated and real datasets, we demonstrate that our methods increase the reproducibility of RNA-seq and small RNA-seq data. Notably, we find that the amount of starting material and sequencing depth, but not the number of PCR cycles, determine PCR duplicate frequency. Finally, we show that computational removal of PCR duplicates based only on their mapping coordinates introduces substantial bias into data analysis.

genomics↗

bioRxiv2017

Pan-arthropod analysis reveals somatic piRNAs as an ancestral TE defence

In animals, small RNA molecules termed PIWI-interacting RNAs (piRNAs) silence transposable elements (TEs), protecting the germline from genomic instability and mutation. piRNAs have been detected in the soma in a few animals, but these are believed to be specific adaptations of individual species. Here, we report that somatic piRNAs were likely present in the ancestral arthropod more than 500 million years ago. Analysis of 20 species across the arthropod phylum suggests that somatic piRNAs targeting TEs and mRNAs are common among arthropods. The presence of an RNA-dependent RNA polymerase in chelicerates (horseshoe crabs, spiders, scorpions) suggests that arthropods originally used a plant-like RNA interference mechanism to silence TEs. Our results call into question the view that the ancestral role of the piRNA pathway was to protect the germline and demonstrate that small RNA silencing pathways have been repurposed for both somatic and germline functions throughout arthropod evolution.

evolutionary biology↗

bioRxiv2017

The genome of Trichoplusia ni, an agricultural pest and novel model for small RNA biology

The cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae), is a destructive insect pest that feeds on a wide range of plants. The High Five cell line (Hi5), originally derived from T. ni ovaries, is often used for efficient expression of recombinant proteins. Here, we report a draft assembly of the 368.2 Mb T. ni genome, with 90.6% of all bases assigned to one of its 28 chromosomes and predicted 14,037 predicted protein-coding genes. Manual curation of gene families involved in chemoreception and detoxification reveals T. ni-specific gene expansions that may explain its widespread distribution and rapid adaptation to insecticides. Using male and female genome sequences, we define Z-linked and repeat-rich W-linked sequences. Transcriptome and small RNA data from T. ni thorax, ovary, testis, and Hi5 cells reveal distinct expression profiles for 295 microRNA- and >393 piRNA-producing loci, as well as 39 genes encoding core small RNA pathway proteins. siRNAs target both endogenous transposons and the exogenous TNCL virus. Surprisingly, T. ni siRNAs are not 2{acute}-O-methylated. Five piRNA-producing loci account for 34.9% piRNAs in the ovary, 49.3% piRNAs in the testis, and 44.0% piRNAs in Hi5 cells. Nearly all of the W chromosome is devoted to piRNA production: >76.0% of bases in the assembled W produce piRNAs in ovary. To enable use of the T. ni germline-derived Hi5 cell line as a model system, we have established efficient genome editing and single-cell cloning protocols. Taken together, the T. ni genome provides insights into pest control and allows Hi5 cells to become a new tool for studying small RNAs ex vivo.

genomics↗