NCRNA

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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 transposon 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.”

EU Research


NCRNA

Non-coding RNA pathways and the mammalian male germline Project Objectives

We propose analysing the contribution of both short and long non-coding RNAs to mammalian male germ cell development and SSC homeostasis. In the mouse male germline the small non-coding piRNAs and their interacting-Piwi proteins Mili and Miwi2 are essential for the establishment of epigenetic transposon silencing. We will determine if Miwi2 expression identifies the illusive SSC population with long-term self-renewal capacity in vivo and explore the biology of this adult stem cell population.

Project Funding The project itself set out to achieve four main goals, the first of which centered around showing that the piRNA pathway does indeed play an important role in repressing transposons in meiotic cells. Researchers have been able to show that it does this by cleaving the transposon messenger RNA. “A piwi-interacting protein called MILI acts almost as a pair of scissors, which is guided to its target by a piRNA. We were able to show that piRNA is the trigger for the molecular scissors. It enables the molecular scissors to cut and neutralise it,” says Professor O’Carroll. The reason this happens in meiosis is that there are big changes to the chromatin landscape at this time, as many different events are affecting chromosomes. “These changes to the chromatin landscape enable certain transposons to become expressed, but these are post-transcription regulators,” explains

in spermatogonial cells. “Its function during germline reprogramming is essential for the spermatogonial stem cells, because if you don’t have normal reprogramming then you have defective gene expression in the spermatagonia,” outlines Professor O’Carroll. The last aim in the project relates to another type of non-coding RNA, called long noncoding RNAs, and how they complicate the germline. “We recently published a paper saying that the germline has a very diverse group of long-coding RNAs, many of which originate from defined classes of transposable elements,” continues Professor O’Carroll. A number of other papers have been published around these four key aims of the project, with the wider goal of building a deeper understanding of the role of the piRNA pathway, beyond its function in germline reprogramming. “We want to understand the contribution of the piRNA

We wanted to understand the function of the piRNA pathway in more detail, beyond its central role in germ-line re-programming. How does it function later on in spermatogenesis – does it regulate transposon

elements? Does it contribute to spermatagonial stem cell biology? Professor O’Carroll. “In the second aim, we asked whether a second Piwi protein called MIWI2 was expressed in a population of spermatagonial stem cells. The answer is that it is indeed expressed.”

Stem cell activity This population of cells can regain stem cell activity, even after damage to the testis, and are very important to the regenerative capacity of an individual in recovering from injury. The third aim in the project centered around investigating why MIWI2 is important

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pathway to spermatogenesis, essentially to ensuring that spermatogenesis actually works,” says Professor O’Carroll. There are many unanswered questions in this respect, so beyond the term of the current project, Professor O’Carroll plans to pursue further research in this area in future. “We understand now that reprogramming is important for latent spermatagonial stem cells. As reprogramming is so important, we would really like to understand the mechanics of reprogramming in more detail,” he says.

ERC-SG-LS3 - ERC Starting Grant - Cellular and Developmental Biology (ERC-2012StG_20111109).

Project Partners

• The University of Edinburgh, United Kingdom (Coordinator) • European Molecular Biology Laboratory, Germany

Contact Details

Project Coordinator, Professor Dónal O’Carroll Chair of Stem Cell Biology Associate Director, MRC Centre for Regenerative Medicine SCRM Building, University of Edinburgh 5 Little France Drive Edinburgh EH16 4UU United Kingdom T: +39 06 900 91222 E: ocarroll@embl.it W: http://www.crm.ed.ac.uk/research/group/ rna-function-germ-and-stem-cell-biology Professor Dónal O’Carroll

Professor Dónal O’Carroll has been Chair of Stem Cell Biology, Head, Institute of Stem Cell Research since 2015. He has also been Associate Director at the MRC Centre for Regenerative Medicine, since 2015. He has been Wellcome Trust Senior Investigator since 2015, ERC Investigator since 2012, and he was Group Leader at EMBL Monterotondo between 2007 and 2015. He has been Adjunct member of faculty at The Rockefeller University since 2007. He was Postdoctoral research at The Rockefeller University, New York between 2001 and 2007. He attained his PhD in 1999, at the Research Institute of Molecular Pathology, in Vienna.

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