The Piwi pathway: from piRNA methylation to histone methylation

M.J. Luteijn

    Research output: ThesisDoctoral thesis 2 (Research NOT UU / Graduation UU)


    Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defense and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. These small RNA molecules play an important role in defending the germline against transposons, which are selfish DNA elements that can move and/or multiply themselves to new positions in the genome, thereby endangering the genomic stability of an organism piRNAs discriminate themselves from most other small RNA molecules by carrying a 2’O methylgroup at their 3’ end. The protein that methylates piRNA is the methyltransferase Hen1. We studied the role of Hen1 in the zebrafish D. rerio and the nematode C. elegans. Zebrafish Hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line. In hen1 mutant testes, piRNAs become unstable and transposons are mildly upregulated. Hen1 protein localizes to nuage through its C-terminal domain, which is a peri-nuclear structure consisting of RNA and many proteins related to RNA-interference. The C. elegans methyltransferase HENN-1 is responsible for the methylation of two germline expressed small RNA species, the 26G siRNA and piRNAs. We show that the stability of piRNAs is hardly affected in henn-1 mutant animals. However, most 26G RNA display reduced stability and have additional uracil and adenines at their 3' end. The affected 26G RNAs are involved in the endogenous RNAi pathway and influence gene expression during zygotic development. However, loss of henn-1 leads only to mild effects on germline expressed genes. In addition, the HENN-1 protein does not form a stable complex with the Piwi protein that bind piRNAs. However, HENN-1 localizes close to the regions that also host Piwi proteins. Small RNAs not only influence expression of proteins at the RNA level, they can also induce silencing at the DNA level by preventing transcription of RNA. We showed that in C. elegans, the Piwi pathway can initiate gene silencing by inducing heterochromatin formation. More specifically, the piRNA pathway initiate an extremely stable form of gene silencing on a transgenic, single-copy target. This type of silencing is faithfully maintained over tens of generations by factors known to act in the nuclear RNAi pathway. This nuclear pathway induces the tri-methylation of lysine 9 of histone 3 - a repressive chromatin mark- on their target loci. In conclusion, we studied two 'methylation steps' of the Piwi-pathway, starting with the 3' end methylation of piRNAs by Hen1 and moving towards the histone methylation initiated by the C elegans specific piRNA-pathway.
    Original languageEnglish
    QualificationDoctor of Philosophy
    Awarding Institution
    • Utrecht University
    • Ketting, R.F., Primary supervisor, External person
    Award date14 May 2013
    Print ISBNs978-90-393-5954-9
    Publication statusPublished - 14 May 2013


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