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

Integrator is recruited to promoter-proximally paused RNA Pol II to generate Caenorhabditis elegans piRNA precursors

Piwi-interacting RNAs (piRNAs) play key roles in germline development and genome defence in metazoans. In C. elegans, piRNAs are transcribed from > 15,000 discrete genomic loci by RNA polymerase II (Pol II), resulting in 28 nt short-capped piRNA precursors. Here, we investigate transcription termination at piRNA loci. We show that the Integrator complex, which terminates snRNA transcription, is recruited to piRNA loci. Moreover, we demonstrate that the catalytic activity of Integrator cleaves nascent capped piRNA precursors associated with promoter-proximal Pol II, resulting in termination of transcription. Loss of Integrator activity, however, does not result in transcriptional readthrough at the majority of piRNA loci. Taken together, our results draw new parallels between snRNA and piRNA biogenesis in nematodes and provide evidence of a role for the Integrator complex as a terminator of promoter-proximal RNA polymerase II during piRNA biogenesis.We would like to thank Juan Cabello for kindly sharing JCP383 ints-6::GFP nematodes with us at the beginning of the study. This work was funded by the United Kingdom Medical Research Council (MC-A652-5PY80 “Epigenetics and Evolution”-PS; MC_UP_1102/1 “Computational Regulatory Genomics”- BL ) and by the Wellcome Trust (106954/Z/15/Z- BL

Loss of the E3 ubiquitin ligases UBR-5 or HECD-1 restores Caenorhabditis elegans development in the absence of SWI/SNF function.

SWItch/sucrose non-fermenting (SWI/SNF) complexes are a family of chromatin remodelers that are conserved across eukaryotes. Mutations in subunits of SWI/SNF cause a multitude of different developmental disorders in humans, most of which have no current treatment options. Here, we identify an alanine-to-valine-causing mutation in the SWI/SNF subunit snfc-5 (SMARCB1 in humans) that prevents embryonic lethality in Caenorhabditis elegans nematodes harboring a loss-of-function mutation in the SWI/SNF subunit swsn-1 (SMARCC1/2 in humans). Furthermore, we found that the combination of this specific mutation in snfc-5 and a loss-of-function mutation in either of the E3 ubiquitin ligases ubr-5 (UBR5 in humans) or hecd-1 (HECTD1 in humans) can restore development to adulthood in swsn-1 loss-of-function mutants that otherwise die as embryos. Using these mutant models, we established a set of 335 genes that are dysregulated in SWI/SNF mutants that arrest their development embryonically but exhibit near wild-type levels of expression in the presence of suppressor mutations that prevent embryonic lethality, suggesting that SWI/SNF promotes development by regulating some subset of these 335 genes. In addition, we show that SWI/SNF protein levels are reduced in swsn-1; snfc-5 double mutants and partly restored to wild-type levels in swsn-1; snfc-5; ubr-5 triple mutants, consistent with a model in which UBR-5 regulates SWI/SNF levels by tagging the complex for proteasomal degradation. Our findings establish a link between two E3 ubiquitin ligases and SWI/SNF function and suggest that UBR5 and HECTD1 could be potential therapeutic targets for the many developmental disorders caused by missense mutations in SWI/SNF subunits