Molecular mechanisms of mammalian germline small RNAs that ensure genome integrity
Final Report Abstract
The main focus of the project supported by the DFG fellowship was the characterization ofa Miwi slicer mutant. Miwi is a Piwi protein expressed in the germ line of mouse. Generally, Piwi proteins are Argonaute proteins representing the core component of the piRNA pathway, a small RNA pathway exclusively acting in the germ line of animals. Slicer designates an RNaseH activity catalyzed by the piwi domain of Argonaute proteins. Substrate recognition is mediated by the bound small RNA, which acts as a guide recruiting the protein to target RNAs via base pairing. Amino acids, which compose the active site (DDH motif) in the piwi domain are also conserved in Miwi suggesting a catalytic activity. Indeed slicer cleavage of target RNAs could be demonstrated in vitro using immune purified Miwi-complexes and in vitro transcribed target RNAs. To investigate to role of Miwi and its slicer activity in vivo we made a mouse mutant where the first amino acid of the active site is changed (ADH). Biochemical assays confirmed that the mutation completely abolish the catalytic activity. The slicer activity of Miwi is essential. Mutant males are sterile caused by a complete, uniform block of spermatogenesis immediately after meiotic division. The search for Miwi target or substrate RNAs, which was performed using different approaches, revealed that LINE 1 retrotransposons are activated in the mutant. This was surprising since transposon repression mainly based on transcriptional silencing by DNA (Cytosine) methylation, which is established already in embryonic (primordial) germ cells. DNA methylation is-not affected in the Miwi slicer mutant. We conclude that the slicer activity of Miwi cleaves LINE 1 transcripts initiating their rapid decay. This is mediated by Miwi bound piRNAs matching to LINE 1 sequences in anti-sense orientation, which represent app. 12% of the piRNAs bound to Miwi. This shows that Miwi has an essential role in the posttranscriptional silencing of transposons during spermatogenesis or spermiogenesis, respectively. Obtained data further provide first indications for the role of piRNAs originating from intergenic regions (pachyten piRNAs).
Publications
- Miwi catalysis is required for piRNA amplification-independent LINE1 transposon silencing. Nature. 2011 Nov 27;480(7376):264-7
Reuter M et al.
