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ABSTRACT In Neurospora, genes not paired during meiosis are targeted by meiotic silencing by unpaired DNA (MSUD). Here, our bimolecular fluorescence complementation (BiFC) study suggests that RNA-directed RNA polymerase, Dicer, Argonaute, and others form a silencing complex in the perinuclear region, with intimate interactions among the majority of them. We have also shown that SAD-2 is likely the anchor for this assembly.
KEYWORDS RNA interference; Neurospora crassa; meiotic silencing by unpaired DNA (MSUD); bimolecular fluorescence complementation (BiFC); fluorescent protein tagging
THE filamentous fungus Neurospora crassa consists of a hyphal network where nuclei and other cellular components share a common cytoplasm. This trait, while beneficial for distributing resources, may promote the spread of detrimental elements such as transposons and viruses. Perhaps for this reason, Neurospora possesses several surveillance mechanisms that operate during different phases of its life cycle (Aramayo and Selker 2013; Billmyre et al. 2013; Nicolás and Ruiz- Vázquez 2013). For example, quelling silences tandem transgenes during vegetative growth (Romano and Macino 1992). Repeat-induced point mutation (RIP), a premeiotic system functioning before nuclear fusion, introduces C/Tmutations to duplicated DNA sequences (Cambareri et al. 1989). Finally, meiotic silencing by unpaired DNA (MSUD) targets genes not paired with a homologous partner during meiosis (Shiu et al. 2001). MSUD begins inside the nucleus when an unpaired gene is detected (Samarajeewa et al. 2014) and acts as a template for the production of an aberrant RNA (aRNA; abnormal RNA that would enter the silencing pathway). A working model for MSUD holds that the aRNA is exported to the perinuclear region, where it is met by a host of RNAirelated proteins, including SAD-1, an RNA-directed RNA polymerase (RdRP) that converts aRNAs into double-strand RNAs (dsRNAs) (Shiu and Metzenberg 2002); SAD-3, a helicase thought to increase SAD-1's processivity on RNA templates (Hammond et al. 2011a); DCL-1, an RNAse III Dicer that cuts dsRNAs into small interfering RNAs (siRNAs) (Alexander et al. 2008); QIP, an exonuclease that converts siRNAs into single strands (Xiao et al. 2010); and SMS-2, an Argonaute protein that uses siRNAs to guide selective destruction of homologous mRNAs (Lee et al. 2003). SAD-2, unlike the others, is not found in other RNA silencing systems but is essential for the perinuclear localization of SAD-1 (Shiu et al. 2006).
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