Rapid evolution and conserved function of the piRNA pathway

Transposons are major genome constituents that can mobilize and trigger mutations, DNA breaks and chromosome rearrangements. Transposon silencing is particularly important in the germline, which is dedicated to transmission of the inherited genome. Piwi-interacting RNAs (piRNAs) guide a host defence system that transcriptionally and post-transcriptionally silences transposons during germline development. While germline control of transposons by the piRNA pathway is conserved, many piRNA pathway genes are evolving rapidly under positive selection, and the piRNA biogenesis machinery shows remarkable phylogenetic diversity. Conservation of core function combined with rapid gene evolution is characteristic of a host-pathogen arms race, suggesting that transposons and the piRNA pathway are engaged in an evolutionary tug of war that is driving divergence of the biogenesis machinery. Recent studies suggest that this process may produce biochemical incompatibilities that contribute to reproductive isolation and species divergence

Biogenesis and germline functions of piRNAs

Small interfering RNAs and microRNAs are generated from double-stranded RNA precursors by the Dicer endonucleases, and function with Argonaute-family proteins to target transcript destruction or to silence translation. A distinct class of 24- to 30-nucleotide-long RNAs, produced by a Dicer-independent mechanism, associates with Piwi-class Argonaute proteins. Studies in flies, fish and mice implicate these Piwi-associated RNAs (piRNAs) in germline development, silencing of selfish DNA elements, and in maintaining germline DNA integrity. However, whether piRNAs primarily control chromatin organization, gene transcription, RNA stability or RNA translation is not well understood, neither is piRNA biogenesis. Here, we review recent studies of piRNA production and function, and discuss unanswered questions about this intriguing new class of small RNAs

TEMP: a computational method for analyzing transposable element polymorphism in populations

Insertions and excisions of transposable elements (TEs) affect both the stability and variability of the genome. Studying the dynamics of transposition at the population level can provide crucial insights into the processes and mechanisms of genome evolution. Pooling genomic materials from multiple individuals followed by high-throughput sequencing is an efficient way of characterizing genomic polymorphisms in a population. Here we describe a novel method named TEMP, specifically designed to detect TE movements present with a wide range of frequencies in a population. By combining the information provided by pair-end reads and split reads, TEMP is able to identify both the presence and absence of TE insertions in genomic DNA sequences derived from heterogeneous samples; accurately estimate the frequencies of transposition events in the population and pinpoint junctions of high frequency transposition events at nucleotide resolution. Simulation data indicate that TEMP outperforms other algorithms such as PoPoolationTE, RetroSeq, VariationHunter and GASVPro. TEMP also performs well on whole-genome human data derived from the 1000 Genomes Project. We applied TEMP to characterize the TE frequencies in a wild Drosophila melanogaster population and study the inheritance patterns of TEs during hybrid dysgenesis. We also identified sequence signatures of TE insertion and possible molecular effects of TE movements, such as altered gene expression and piRNA production. TEMP is freely available at github: https://github.com/JialiUMassWengLab/TEMP.git. Acids Research

Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection

BACKGROUND: High throughput DNA sequencing technology has enabled quantification of all the RNAs in a cell or tissue, a method widely known as RNA sequencing (RNA-Seq). However, non-coding RNAs such as rRNA are highly abundant and can consume \u3e70% of sequencing reads. A common approach is to extract only polyadenylated mRNA; however, such approaches are blind to RNAs with short or no poly(A) tails, leading to an incomplete view of the transcriptome. Another challenge of preparing RNA-Seq libraries is to preserve the strand information of the RNAs. DESIGN: Here, we describe a procedure for preparing RNA-Seq libraries from 1 to 4 mug total RNA without poly(A) selection. Our method combines the deoxyuridine triphosphate (dUTP)/uracil-DNA glycosylase (UDG) strategy to achieve strand specificity with AMPure XP magnetic beads to perform size selection. Together, these steps eliminate gel purification, allowing a library to be made in less than two days. We barcode each library during the final PCR amplification step, allowing several samples to be sequenced in a single lane without sacrificing read length. Libraries prepared using this protocol are compatible with Illumina GAII, GAIIx and HiSeq 2000 platforms. DISCUSSION: The RNA-Seq protocol described here yields strand-specific transcriptome libraries without poly(A) selection, which provide approximately 90% mappable sequences. Typically, more than 85% of mapped reads correspond to protein-coding genes and only 6% derive from non-coding RNAs. The protocol has been used to measure RNA transcript identity and abundance in tissues from flies, mice, rats, chickens, and frogs, demonstrating its general applicability

Distinct Functions for the Drosophila piRNA Pathway in Genome Maintenance and Telomere Protection

Transposons and other selfish DNA elements can be found in all phyla, and mobilization of these elements can compromise genome integrity. The piRNA (PIWI-interacting RNA) pathway silences transposons in the germline, but it is unclear if this pathway has additional functions during development. Here we show that mutations in the Drosophila piRNA pathway genes, armi, aub, ago3, and rhi, lead to extensive fragmentation of the zygotic genome during the cleavage stage of embryonic divisions. Additionally, aub and armi show defects in telomere resolution during meiosis and the cleavage divisions; and mutations in lig-IV, which disrupt non-homologous end joining, suppress these fusions. By contrast, lig-IV mutations enhance chromosome fragmentation. Chromatin immunoprecipitation studies show that aub and armi mutations disrupt telomere binding of HOAP, which is a component of the telomere protection complex, and reduce expression of a subpopulation of 19- to 22-nt telomere-specific piRNAs. Mutations in rhi and ago3, by contrast, do not block HOAP binding or production of these piRNAs. These findings uncover genetically separable functions for the Drosophila piRNA pathway. The aub, armi, rhi, and ago3 genes silence transposons and maintain chromosome integrity during cleavage-stage embryonic divisions. However, the aub and armi genes have an additional function in assembly of the telomere protection complex

Adaptive Evolution Targets a piRNA Precursor Transcription Network

In Drosophila, transposon-silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, which are bound by the Rhino-Deadlock-Cutoff complex. The HP1 homolog Rhino binds to Deadlock, which recruits TRF2 to promote non-canonical transcription from both genomic strands. Cuff function is less well understood, but this Rai1 homolog shows hallmarks of adaptive evolution, which can remodel functional interactions within host defense systems. Supporting this hypothesis, Drosophila simulans Cutoff is a dominant-negative allele when expressed in Drosophila melanogaster, in which it traps Deadlock, TRF2, and the conserved transcriptional co-repressor CtBP in stable complexes. Cutoff functions with Rhino and Deadlock to drive non-canonical transcription. In contrast, CtBP suppresses canonical transcription of transposons and promoters flanking the major germline clusters, and canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution thus targets interactions among Cutoff, TRF2, and CtBP that balance canonical and non-canonical piRNA precursor transcription

Adaptive evolution targets a piRNA precursor transcription network [preprint]

In Drosophila, transposon-silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, which are transcribed from internal non-canonical initiation sites and flanking canonical promoters. Rhino binds to Deadlock, which recruits TRF2 to promote non-canonical transcription of these loci. Cuff co-localizes with Rhino and Del. The role of Cuff is less well understood, but the cuff gene shows hallmarks of adaptive evolution, which frequently targets functional interactions within host defense systems. We show that Drosophila simulans cuff is a dominant negative allele when expressed in Drosophila melanogaster, where it traps Deadlock, TRF2 and the transcriptional co-repressor CtBP in stable nuclear complexes. Cuff promotes Rhino and Deadlock localization, driving non-canonical transcription. CtBP, by contrast, suppresses canonical cluster and transposon transcription, which interferes with downstream non-canonical transcription and piRNA production. Cuff, TRF2 and CtBP thus form a network that balances canonical and non-canonical piRNA precursor transcription

Somatic piRNAs and Transposons are Differentially Regulated During Skeletal Muscle Atrophy and Programmed Cell Death [preprint]

PiWi-interacting RNAs (piRNAs) are small single-stranded RNAs that can repress transposon expression via epigenetic silencing and transcript degradation. They have been identified predominantly in the ovary and testis, where they serve essential roles in transposon silencing in order to protect the integrity of the genome in the germline. The potential expression of piRNAs in somatic cells has been controversial. In the present study we demonstrate the expression of piRNAs derived from both genic and transposon RNAs in the intersegmental muscles (ISMs) from the tobacco hawkmoth Manduca sexta. These piRNAs are abundantly expressed, are ~27 nt long, map antisense to transposons, are oxidation resistant, exhibit a uridine bias at their first nucleotide, and amplify via the canonical ping-pong pathway. An RNA-seq analysis demonstrated that 20 piRNA pathway genes are expressed in the ISMs and are developmentally regulated. The abundance of piRNAs does not change when the muscles initiate developmentally-regulated atrophy, but are repressed when cells become committed to undergo programmed cell death at the end of metamorphosis. This change in piRNA expression is associated with the targeted repression of several retrotransposons and the induction of specific DNA transposons. The developmental changes in the expression of piRNAs, piRNA pathway genes, and transposons are all regulated by 20-hydroxyecdysone, the steroid hormone that controls the timing of ISM death. Taken together, these data provide compelling evidence for the existence of piRNA in somatic tissues and suggest that they may play roles in developmental processes such as programmed cell death

Somatic piRNAs and Transposons are Differentially Expressed Coincident with Skeletal Muscle Atrophy and Programmed Cell Death

PIWI-interacting RNAs (piRNAs) are small single-stranded RNAs that can repress transposon expression via epigenetic silencing and transcript degradation. They have been identified predominantly in the ovary and testis, where they serve essential roles in transposon silencing in order to protect the integrity of the genome in the germline. The potential expression of piRNAs in somatic cells has been controversial. In the present study we demonstrate the expression of piRNAs derived from both genic and transposon RNAs in the intersegmental muscles (ISMs) from the tobacco hawkmoth Manduca sexta. These piRNAs are abundantly expressed, ∼27 nt long, map antisense to transposons, are oxidation resistant, exhibit a 5’ uridine bias, and amplify via the canonical ping-pong pathway. An RNA-seq analysis demonstrated that 19 piRNA pathway genes are expressed in the ISMs and are developmentally regulated. The abundance of piRNAs does not change when the muscles initiate developmentally-regulated atrophy, but are repressed coincident with the commitment of the muscles undergo programmed cell death at the end of metamorphosis. This change in piRNA expression is correlated with the repression of several retrotransposons and the induction of specific DNA transposons. The developmentally-regulated changes in the expression of piRNAs, piRNA pathway genes, and transposons are all regulated by 20-hydroxyecdysone, the steroid hormone that controls the timing of ISM death. Taken together, these data provide compelling evidence for the existence of piRNA in somatic tissues and suggest that they may play roles in developmental processes such as programmed cell death

A benchmark and an algorithm for detecting germline transposon insertions and measuring de novo transposon insertion frequencies

Transposons are genomic parasites, and their new insertions can cause instability and spur the evolution of their host genomes. Rapid accumulation of short-read whole-genome sequencing data provides a great opportunity for studying new transposon insertions and their impacts on the host genome. Although many algorithms are available for detecting transposon insertions, the task remains challenging and existing tools are not designed for identifying de novo insertions. Here, we present a new benchmark fly dataset based on PacBio long-read sequencing and a new method TEMP2 for detecting germline insertions and measuring de novo \u27singleton\u27 insertion frequencies in eukaryotic genomes. TEMP2 achieves high sensitivity and precision for detecting germline insertions when compared with existing tools using both simulated data in fly and experimental data in fly and human. Furthermore, TEMP2 can accurately assess the frequencies of de novo transposon insertions even with high levels of chimeric reads in simulated datasets; such chimeric reads often occur during the construction of short-read sequencing libraries. By applying TEMP2 to published data on hybrid dysgenic flies inflicted by de-repressed P-elements, we confirmed the continuous new insertions of P-elements in dysgenic offspring before they regain piRNAs for P-element repression. TEMP2 is freely available at Github: https://github.com/weng-lab/TEMP2