Following the piRNA pathway to genomic stability The piRNA pathway is known to play a central role in germline reprogramming, now researchers in the NCRNA project aim to investigate its function in other areas, including spermatogenesis and transposon silencing. We spoke to Professor Dónal O’Carroll about the project’s work in using cutting-edge techniques to gain new insights into the role of the piRNA pathway A
core responsibility of the mammalian germline is the transmission of the genome from one generation to the next. One of the major challenges in maintaining the integrity of the germline is to ensure that transposons, DNA sequences that constitute between 4050 percent of the mammalian genome, do not cause too many de novo mutations, as Professor Dónal O’Carroll explains. “If these transposons are transcribed they can effectively copy and paste themselves into novel locations in the genome, and that creates genetic damage,” he outlines. Based at the University of Edinburgh’s Centre for Regenerative Medicine, Professor O’Carroll investigates fundamental questions around both short and long non-coding RNAs and the mammalian germline. “The germline is derived from somatic cells. They’ve got two sets of chromosomes – one maternal, one paternal – and those chromosomes are not epigenetically equal,” he continues. “During mammalian development there’s an event called germline reprogramming, where all the epigenetic information and all the DNA methylation are erased, and they have to be put back.”
Germline reprogramming There is a window during germline reprogramming when some transposons may become active, and unless they’re suppressed or targeted in some way this
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could cause de novo integrations and mutations. This is where the piRNA pathway (piwi-interacting RNA), which relates to a class of small, non-coding RNA molecules, comes in. “The classical function of the piRNA pathway is to repress transposons. As it represses transposons during germline reprogramming, it also signals for them to be de novo DNA methylated, to silence them later in life,” explains Professor O’Carroll. This topic has attracted a lot
Histological section of a mouse seminiferous tubule. The most undifferentiated germ cells are found in the basal (outer) part of the tubule that differentiate as they progress towards the lumen (centre) where the nascent sperm cells are visible.
of research attention over the years, yet there is much still to learn about the wider role of the piRNA pathway, now Professor O’Carroll and his colleagues aim to shed further light on the topic in an EC-backed project. “We wanted to understand the function of the piRNA pathway in more detail, beyond its central role in germ-line
reprogramming. How does it function later on in spermatogenesis – does it regulate transposable elements? Does it contribute to spermatagonial stem cell biology?” he asks. Researchers have been applying conditional genetics methods in this work, using engineered point mutations to make catalytically inactive proteins and gain new insights into the underlying mechanisms behind spermatogenesis, the process by which mammals produce sperm cells. These techniques are being applied on mice, with researchers investigating how transposons are silenced. “We can delete genes, and we can change and restructure their function in vivo by removing active enzymes,” outlines Professor O’Carroll. A wide variety of other techniques are also being applied in the project, which allows Professor O’Carroll and his colleagues to investigate further key questions around the piRNA pathway. “In our laboratory we essentially couple genetics with high-throughput sequencing from very defined populations of cells. We’re looking at cells from the testis,” he continues. “The genetics techniques allow you to test a particular hypothesis around the role of a gene. So if a gene is important, then the sequencing will give you more detail. The sequencing in other experiments will then help you understand the mechanism by which the gene is important.”
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