Germ plasm condensates and the PIWI pathway: maternal and zygotic elements that shape the germline

TY - THES

T1 - Germ plasm condensates and the PIWI pathway: maternal and zygotic elements that shape the germline

AU - Roovers, Elke F

PY - 2019/4/24

Y1 - 2019/4/24

N2 - Sexually reproducing organisms produce germ cells in order to pass on their genetic information to the next generation. These cells require specialization during early development and in addition, the protection by the PIWI pathway: a germline-specific small RNA pathway that silences transposable elements through the recognition of their transcripts by PIWI-interacting RNAs (piRNAs) bound to a PIWI protein. In this thesis, several aspects of these processes are studied in order to broaden our knowledge regarding these matters. Chapter 1 provides a general overview of germline development and the PIWI pathway in different model organisms. Furthermore, I will discuss a process called liquid-liquid phase separation, which plays a role in these processes as well, but also in many other biological phenomena and, when out of control, various disease phenotypes including many neurological illnesses. In chapter 2, we investigate one aspect of piRNA biogenesis in more detail: its 3’ end processing. This step is catalyzed by a 3’-to-5’ exonuclease, ‘Trimmer,’ which requires recruitment to the precursor by a Tudor domain-containing protein TdrKH. In some organisms such as mouse, TdrKH is therefore an essential factor for fertility of the animal. Using CRISPR-Cas9, we obtained tdrkh mutant zebrafish and observe that, even though piRNAs are longer, likely reflecting 3’ end processing defects, we still see that part of the population remains fertile. We will speculate about the possible reasons for this, including the shift to a different arm of the pathway. In mammals, the PIWI pathway has been studied mostly in the male germline. The reason for this is the absence of an essential PIWI pathway in the female germline of the mouse. It has therefore been assumed that the PIWI pathway is dispensable in the female mammalian germline. The recent discovery of an overly active Dicer variant in the murine ovary and the evolutionary loss of a PIWI paralog, PIWIL3, prompted us to explore a potential role for PIWIL3 and piRNAs in the female germline of other mammalian species. Our results, described in chapter 3, suggest that the PIWI pathway does play a role in female germline of other mammals, including humans. In chapter 4 and 5 we investigate properties of germ cell-specific subcellular aggregates formed through liquid-liquid phase separation. The protein Bucky ball (Buc) contains a prion-like domain and is an essential component of the Balbiani body, an example of such an aggregate in the oocyte. This structure is highly dynamic throughout oogenesis and embryogenesis. One factor that modulates Buc-containing structures is Tdrd6a, which increases the mobility of these structures, which is ultimately required for its normal function in germ cell specification. This is described in chapter 4. In chapter 5 we follow up on a mutated version of Buc, Buc-RtoK, which in the absence of a wild-type copy of Buc, fails to form a Balbiani body. We find that, without the presence of a Balbiani body, Buc-RtoK can still phase-separate during the late vegetal pathway. Furthermore, we identify novel candidates that co-localize and affect Buc-structures in BmN4 cells.

AB - Sexually reproducing organisms produce germ cells in order to pass on their genetic information to the next generation. These cells require specialization during early development and in addition, the protection by the PIWI pathway: a germline-specific small RNA pathway that silences transposable elements through the recognition of their transcripts by PIWI-interacting RNAs (piRNAs) bound to a PIWI protein. In this thesis, several aspects of these processes are studied in order to broaden our knowledge regarding these matters. Chapter 1 provides a general overview of germline development and the PIWI pathway in different model organisms. Furthermore, I will discuss a process called liquid-liquid phase separation, which plays a role in these processes as well, but also in many other biological phenomena and, when out of control, various disease phenotypes including many neurological illnesses. In chapter 2, we investigate one aspect of piRNA biogenesis in more detail: its 3’ end processing. This step is catalyzed by a 3’-to-5’ exonuclease, ‘Trimmer,’ which requires recruitment to the precursor by a Tudor domain-containing protein TdrKH. In some organisms such as mouse, TdrKH is therefore an essential factor for fertility of the animal. Using CRISPR-Cas9, we obtained tdrkh mutant zebrafish and observe that, even though piRNAs are longer, likely reflecting 3’ end processing defects, we still see that part of the population remains fertile. We will speculate about the possible reasons for this, including the shift to a different arm of the pathway. In mammals, the PIWI pathway has been studied mostly in the male germline. The reason for this is the absence of an essential PIWI pathway in the female germline of the mouse. It has therefore been assumed that the PIWI pathway is dispensable in the female mammalian germline. The recent discovery of an overly active Dicer variant in the murine ovary and the evolutionary loss of a PIWI paralog, PIWIL3, prompted us to explore a potential role for PIWIL3 and piRNAs in the female germline of other mammalian species. Our results, described in chapter 3, suggest that the PIWI pathway does play a role in female germline of other mammals, including humans. In chapter 4 and 5 we investigate properties of germ cell-specific subcellular aggregates formed through liquid-liquid phase separation. The protein Bucky ball (Buc) contains a prion-like domain and is an essential component of the Balbiani body, an example of such an aggregate in the oocyte. This structure is highly dynamic throughout oogenesis and embryogenesis. One factor that modulates Buc-containing structures is Tdrd6a, which increases the mobility of these structures, which is ultimately required for its normal function in germ cell specification. This is described in chapter 4. In chapter 5 we follow up on a mutated version of Buc, Buc-RtoK, which in the absence of a wild-type copy of Buc, fails to form a Balbiani body. We find that, without the presence of a Balbiani body, Buc-RtoK can still phase-separate during the late vegetal pathway. Furthermore, we identify novel candidates that co-localize and affect Buc-structures in BmN4 cells.

KW - Germline development

KW - PIWI pathway

KW - piRNA

KW - phase separation

KW - Bucky Ball

KW - Tdrd6a

M3 - PhD thesis

SN - 978-94-6182-948-1

PB - Utrecht University

ER -