The Trajectory of KoRV-A Evolution Indicates Initial Integration into the Koala Germline Genome Near Coffs Harbour.

Background: Koala Retrovirus-A is a gamma-retrovirus that is spreading across wild koala populations through horizontal and vertical transmission, contributing significantly to genomic diversity across and even within koala populations. Previous studies have estimated that KoRV-A initially integrated into the koala genome less than 50,000 years ago, but the precise origins and the patterns of spread after its endogenization remain unclear.

Results: In this study, we analyzed germline insertions of KoRV-A using whole-genome sequencing data from 405 wild koalas, representing nearly the species' entire geographic range.

The piRNA Response to Retroviral Invasion of the Koala Genome.

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased.

UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery.

piRNAs silence transposons during germline development. In Drosophila, transcripts from heterochromatic clusters are processed into primary piRNAs in the perinuclear nuage. The nuclear DEAD box protein UAP56 has been previously implicated in mRNA splicing and export, whereas the DEAD box protein Vasa has an established role in piRNA production and localizes to nuage with the piRNA binding PIWI proteins Ago3 and Aub.

Adaptation to P element transposon invasion in Drosophila melanogaster.

Transposons evolve rapidly and can mobilize and trigger genetic instability. Piwi-interacting RNAs (piRNAs) silence these genome pathogens, but it is unclear how the piRNA pathway adapts to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo.

Heterotypic piRNA Ping-Pong requires qin, a protein with both E3 ligase and Tudor domains.

piRNAs guide PIWI proteins to silence transposons in animal germ cells. Reciprocal cycles of piRNA-directed RNA cleavage--catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila melanogaster--expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate.

The Drosophila HP1 homolog Rhino is required for transposon silencing and piRNA production by dual-strand clusters.

Piwi-interacting RNAs (piRNAs) silence transposons and maintain genome integrity during germline development. In Drosophila, transposon-rich heterochromatic clusters encode piRNAs either on both genomic strands (dual-strand clusters) or predominantly one genomic strand (uni-strand clusters). Primary piRNAs derived from these clusters are proposed to drive a ping-pong amplification cycle catalyzed by proteins that localize to the perinuclear nuage.

DNA damage-induced cell death is enhanced by progression through mitosis.

Progression through the G(2)/M transition following DNA damage is linked to cytokinesis failure and mitotic death. In four different transformed cell lines and two human embryonic stem cell lines, we find that DNA damage triggers mitotic chromatin decondensation and global phosphorylation of histone H2AX, which has been associated with apoptosis. However, extended time-lapse studies in HCT116 colorectal cancer cells indicate that death does not take place during mitosis, but 72% of cells die within 3 days of mitotic exit.

grp (chk1) replication-checkpoint mutations and DNA damage trigger a Chk2-dependent block at the Drosophila midblastula transition.

The 13 syncytial cleavage divisions that initiate Drosophila embryogenesis are under maternal genetic control. The switch to zygotic regulation of development at the midblastula transition (MBT) follows mitosis 13, when the cleavage divisions terminate, transcription increases and the blastoderm cellularizes. Embryos mutant for grp, which encodes Checkpoint kinase 1 (Chk1), are DNA-replication-checkpoint defective and fail to cellularize, gastrulate or to initiate high-level zygotic transcription at the MBT.

Drosophila rasiRNA pathway mutations disrupt embryonic axis specification through activation of an ATR/Chk2 DNA damage response.

Small repeat-associated siRNAs (rasiRNAs) mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila rasiRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization as well as asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellate silencing.

RISC assembly defects in the Drosophila RNAi mutant armitage.

The putative RNA helicase, Armitage (Armi), is required to repress oskar translation in Drosophila oocytes; armi mutant females are sterile and armi mutations disrupt anteroposterior and dorsoventral patterning. Here, we show that armi is required for RNAi. armi mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro.

The Drosophila SDE3 homolog armitage is required for oskar mRNA silencing and embryonic axis specification.

Polarization of the microtubule cytoskeleton during early oogenesis is required to specify the posterior of the Drosophila oocyte, which is essential for asymmetric mRNA localization during mid-oogenesis and for embryonic axis specification. The posterior determinant oskar mRNA is translationally silent until mid-oogenesis. We show that mutations in armitage and three components of the RNAi pathway disrupt oskar mRNA translational silencing, polarization of the microtubule cytoskeleton, and posterior localization of oskar mRNA.

Kinesin I-dependent cortical exclusion restricts pole plasm to the oocyte posterior.

Microtubules and the plus-end-directed microtubule motor Kinesin I are required for the selective accumulation of oskar mRNA at the posterior cortex of the Drosophila melanogaster oocyte, which is essential to posterior patterning and pole plasm assembly. We present evidence that microtubule minus ends associate with the entire cortex, and that Kinesin and microtubules are not required for oskar mRNA association with the posterior pole, but prevent ectopic localization of this transcript and the pole plasm proteins Oskar and Vasa to other cortical regions. Cortical binding of oskar mRNA seems to be dependent on the actin cytoskeleton.