Biology subjects

Zamore, P. D.

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

11 recordsLinked to original sources

bioRxiv2025

Piwi and piRNAs repress transcription of aberrant rRNA genes containing retrotransposon fragments

The genomes of eukaryotes comprise clusters of repeated rRNA genes, including defective copies that are silenced by poorly understood mechanisms. In Drosophila melanogaster, many 28S rDNA genes contain insertions of R1 or R2 retrotransposons. R2 elements can only transcribe as part of the pre-rRNA and then excise by the R2 ribozyme. Some rRNA genes carry truncated insertions, creating defective rRNA from which the R2 sequence cannot be excised. Here, we report that in Drosophila ovaries, the nuclear protein Piwi loaded with PIWI-interacting RNA (piRNA) is required to repress transcription of rDNA units with extensively truncated R2 insertions (R2short). Without Piwi, R2short-rDNA generates stable aberrant 28S rRNA containing [~]200 nt of R2 sequence. These rRNAs are excluded from cytoplasmic ribosomes and instead accumulate within the nucleoli of germ cells, where they may disrupt nucleolar homeostasis. Overall, our results show the involvement of the piRNA pathway in quality control of ribosomal transcription.

molecular biology↗

bioRxiv2022

Relaxed targeting rules allow PIWI-clade Argonaute proteins to silence ever-mutating transposons

In animals, piRNAs direct PIWI-clade Argonaute proteins to slice complementary transposon transcripts. Transposons can evade silencing through target site mutations. We report that PIWIs efficiently cleave transcripts only partially paired to their piRNA guide. Measurements of mouse PIWI protein affinity and cleavage rates for thousands of RNAs in vitro and in vivo show that PIWI slicing tolerates mismatches to any target nucleotide, including those flanking the scissile phosphate. Although piRNA 5 terminal nucleotides accelerate target finding, they are dispensable for binding or catalysis--unlike AGO-clade Argonautes, which require uninterrupted siRNA:target pairing from the seed to the nucleotides past the scissile bond. PIWIs are thus better equipped than AGOs to target newly acquired or rapidly diverging endogenous transposons without recourse to novel small RNA guides.

molecular biology↗

bioRxiv2022

Structural Basis of MicroRNA Biogenesis by Dicer-1 and Its Partner Protein Loqs-PB

In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 and its partner Loqs-PB. The structures show Dicer-1*Loqs-PB (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer-1*Loqs-PB heterodimer, enabling conformational proofreading. The Dicer-1 dsRBD and three Loqs-PB dsRBD domains form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-mRNA cleavage shifts the RNA- binding domains and tightens Dicer-1, promoting product release. Our data suggest a model for how the Dicer-1*Loqs-PB complex effects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and sequential product release.

molecular biology↗

bioRxiv2021

The tiny, conserved zinc-finger protein GTSF1helps PIWI proteins achieve their full catalytic potential

Argonaute proteins use nucleic acid guides to find and bind specific DNA or RNA target sequences. Argonaute proteins can be found in all kingdoms of life, and play diverse biological functions including genome defense, gene regulation, and chromosome partitioning. Many Argonautes retain their ancestral endoribonuclease activity, cleaving the phosphodiester bond between target nucleotides t10 and t11. In animals, a specialized class of Argonautes, the PIWI proteins, use 21-35 nt PIWI-interacting RNAs (piRNAs) to direct transposon silencing, protect the germline genome, and regulate gene expression during gametogenesis1. The piRNA pathway is required for fertility in one or both sexes of nearly all animals. Both piRNA production and function require RNA cleavage catalyzed by PIWI proteins. Spermatogenesis in mice and other placental mammals requires three distinct, developmentally regulated PIWI proteins: MIWI (PIWIL1), MILI (PIWIL2), and MIWI2 (PIWIL4)2-4. The piRNA-guided endoribonuclease activities of MIWI and MILI are essential to produce functional sperm5, 6. piRNA-directed silencing in mice and insects also requires Gametocyte-Specific Factor 1 (GTSF1), a PIWI-associated protein of unknown function7-12. Here, we report that GTSF1 potentiates the weak, intrinsic, piRNA-directed RNA cleavage activities of PIWI proteins, transforming them into efficient endoribonucleases. GTSF1 represents the first example of an auxiliary protein that potentiates the catalytic activity of an Argonaute protein.

biochemistry↗

bioRxiv2020

Terminal Modification, Sequence, and Length Determine Small RNA Stability in Animals

In animals, piRNAs, siRNAs, and miRNAs silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3' terminal trimming and 2'-O-methylation. Both trimming and methylation influence piRNA stability. Here, we report that trimming and methylation protect mouse piRNAs from different decay mechanisms. In the absence of 2'-O-methylation, mouse piRNAs with extensive complementarity to long RNAs become unstable. In flies, 2'-O-methylation similarly protects both piRNAs and siRNAs from complementarity-dependent destabilization. Animal miRNAs are unmethylated, and complementarity-dependent destabilization helps explain differences in miRNA decay rates in both mice and flies. In contrast, trimming protects mouse piRNAs from a separate degradation pathway unaffected by target complementarity but sensitive to the 3' terminal, untrimmed sequence. Because distinct sets of mouse piRNAs are protected by each of these mechanisms, loss of both trimming and 2'-O-methylation causes the piRNA pathway to collapse, demonstrating that these two small RNA modifications collaborate to stabilize piRNAs. HighlightsO_LI2'-O-methylation protects mouse and fly piRNAs from complementarity-dependent decay C_LIO_LI2'-O-methylation protects fly siRNAs with extensive complementarity to long RNAs C_LIO_LIComplementarity to long RNAs predicts the half-life of fly and mouse miRNAs C_LIO_LIMouse pre-piRNA decay reflects both pre-piRNA sequence and PIWI protein identity C_LI

molecular biology↗

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↗