Referentes narrativos del sentido de la vida en la historia personal y social.

The second clade of PPD proteins consists of homologues of the Drosophila P- element induced wimpy testis protein - Piwi. First identified in 1997,

constitutional knockout studies showed that Piwi mutants had reduced numbers of adult germ cells, despite normal numbers of embryonic germline stem cells (Lin and Spradling 1997). It has subsequently been shown that this protein is essential for the maintenance of germline stem cells (Cox, Chao et al. 1998), and that this function is likely to be reliant on their role in suppressing the expression of retrotransposons, thereby protecting from integration associated mutational events (Aravin, Hannon et al. 2007).

1.3.3.1 PIWI clade proteins in mammals

Piwi homology has been described in a number of mammals. Amongst these are the murine Miwi (mouse Piwi), Mili (Miwi like) (Kuramochi-Miyagawa,

Kimura et al. 2001; Deng and Lin 2002) and Miwi2 (Carmell, Girard et al. 2007), and their human homologues PIWIL1 (PIWI-Like 1, HIWI) , PIWIL2 (HILI), PIWIL4 (HIWI2) and the additional PIWIL3 (Sharma, Nelson et al. 2001; Sasaki, Shiohama et al. 2003). The most extensively studied amongst these are the murine homologues which, as in flies, are expressed in a developmentally controlled manner in testes (but not in ovaries) (Aravin, Sachidanandam et al. 2007). Knockout models again show varying blocks in spermatogenesis without an effect on testicular primordial germ cell numbers or ovarian gametogenesis (Deng and Lin 2002; Kuramochi-Miyagawa, Kimura et al. 2004; Carmell, Girard et al. 2007).

Analysis of the expression of PIWI-Like genes in humans is less thorough. Expression of PIWIL4 seems to be ubiquitous (Sugimoto, Kage et al. 2007), whilst expression of PIWIL2 and 3 is largely restricted to the testis (Sasaki, Shiohama et al. 2003). The best studied family member, at least in health, is PIWIL1. Within the testis, expression is restricted to germline cells,

spermatocytes and spermatids (Qiao, Zeeman et al. 2002), but expression has also been demonstrated in heart, brain, kidney, pancreas, prostate, skeletal

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muscle and CD34+ haematopoietic precursors, where expression is lost during in vitro differentiation culture (Sharma, Nelson et al. 2001).

1.3.3.2 Mammalian PIWI interacting RNAs - piRNAs

In 2006, a novel class of small RNAs was found to associate with PIWI clade proteins in both flies (Saito, Nishida et al. 2006; Vagin, Sigova et al. 2006) and mammals (Aravin, Gaidatzis et al. 2006; Girard, Sachidanandam et al. 2006; Grivna, Beyret et al. 2006; Lau, Seto et al. 2006; Watanabe, Takeda et al. 2006), the Piwi-interacting RNA (piRNA). Whilst in flies a heavy bias towards sequences derived from repeat elements supported a primary role in control of retrotransposons, mammalian piRNAs showed less specificity for repeat

elements, suggesting more diverse mechanisms of action in mammals. Interestingly, the balance of sequences derived from repeat elements versus exonic elements varies during murine embryogenesis, suggesting also a dynamic function for these complexes (Aravin, Sachidanandam et al. 2007; Gan, Lin et al. 2011).

Tens of thousands of piRNA sequences are transcribed from a relatively limited number (50-100) of piRNA clusters (Aravin, Gaidatzis et al. 2006; Girard,

Sachidanandam et al. 2006; Grivna, Beyret et al. 2006; Lau, Seto et al. 2006; Watanabe, Takeda et al. 2006). They are believed to be processed from long (25-35 kilobases) single stranded RNA precursors (Betel, Sheridan et al. 2007) by an unidentified endonuclease and 2’-O-methylated at the 3’ end by HEN1 (Kirino and Mourelatos 2007). This methylation both stabilises piRNAs and contributes to their PIWI protein binding specificity (Tian, Simanshu et al. 2011).

1.3.4 Non-canonical RNAi pathways – PIWI/piRNAs functions

The original view of Piwi function in Drosophila germline stem cells was that of retrotransposon control. Whilst a novel mechanism is thought to underlie piRNA generation, known as the Ping-Pong cycle, the subsequent effector function is believed to be similar to canonical RNAi mediated by miRNAs, namely post-transcriptional mRNA degradation. As PIWI clade proteins have conserved RNase III domains it seems likely that this remains an important function in mammalian homologues of Piwi, but increasing evidence points towards additional functions of the PIWI/piRNA complex in mammals. The most work on non-canonical PIWI/piRNA function exists in the field of methylation. Mammalian PIWI proteins are expressed during embryogenesis, and in particular during the period of de novo DNA methylation of embryonic germ cells (day 14.5 post coitum – day 2/3 after birth). This is a critical period for establishing methylation of transposons in the male germline, preventing the subsequent expression of these genes. Both Mili and Miwi knockout mice have reduced testis specific CpG methylation of retrotransposons and an associated increase in retrotransposon expression, a phenotype similar to that of DNA methylation deficient dnmt3L knockout animals (Aravin, Sachidanandam et al. 2007; Carmell, Girard et al. 2007; Kuramochi-Miyagawa, Watanabe et al. 2008). Furthermore, a role for piRNAs in the maintenance of paternal imprinting has been described (Watanabe, Tomizawa et al. 2011).

Whilst the vast majority of studies have concentrated on the function of PIWI proteins in the control of retrotransposons in germline specific stem cells, a recent study in Aplysia, the sea hare, has demonstrated a quite different function for PIWI/piRNA complexes. Thomas Tuschl’s and Eric Kandel’s laboratories identified piRNAs in small RNA libraries produced from Aplysia neurones (Rajasethupathy, Antonov et al. 2012). PIWI/piRNA complexes

modulated the expression of the transcriptional repressor CREB2 in response to the neuromodulatory agent serotonin, known to be involved in learning and memory. This effect was mediated by a sequence specific methylation of the CREB2 promoter. This study provides the first example of the effect of

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PIWI/piRNA directed CpG methylation on gene function, in addition to which it is an extragonadal effect.

A small number of less comprehensive studies have also suggested possible roles for PIWI proteins in maintaining histone modifications, both in mice (Wang, Han et al. 2011) and humans (Sugimoto, Kage et al. 2007).