Characterization of Argonaute-associated proteins in meiotic silencing by unpaired DNA
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[EMBARGOED UNTIL 08/01/2026] Neurospora crassa, a filamentous fungus, employs a post-transcriptional gene silencing (PTGS) mechanism known as meiotic silencing by unpaired DNA (MSUD) to protect its genome during meiosis. This process, which relies on RNA interference (RNAi)-related proteins, targets and silences genes lacking a homologous pairing partner during prophase I. While some of these proteins function within the nucleus or shuttle between the nucleus and cytoplasm, many are localized in the perinuclear region, where MSUD activity is highly concentrated. MSUD targets genetic parasites like transposable elements and viruses. When such a parasite generates an unpaired DNA segment during homologous pairing, an RNA signal is transcribed and exported from the nucleus. In the perinuclear region, this aberrant RNA is processed by an RNAi protein complex into short-interfering RNAs (siRNAs), which then guide the suppressor of meiotic silencing-2 (SMS-2) Argonaute to degrade homologous mRNAs. In Neurospora, the suppressor of ascus dominance-9 (SAD-9) protein is homologous to the DEAD-box helicases Belle and Vasa in Drosophila. DEADbox helicases are known to recruit Argonaute proteins in certain organisms. Deletion mutants of sad-9 are highly impaired in meiotic silencing. Localization experiment shows that SAD-9 functions to recruit the SMS-2 Argonaute to the perinuclear region during MSUD. While the absence of SAD-9 does not appear to impact vegetative growth and development, it severely impairs sexual reproduction. The heat shock protein 70 (HSP70) family is a highly conserved group of chaperone proteins found in virtually all organisms. Within the cell, HSP70 performs diverse functions, including protein folding, responding to stress, and facilitating protein translocation to organelles. HSP70 has also been implicated in RNAi processes. In Drosophila, an HSP70 protein has been shown to interact with Argonaute, altering its conformation to enable the loading of siRNAs. Several HSP-70 proteins exist in Neurospora, and HSP70-1 mediates meiotic silencing. HPS70-1 interacts with the SMS-2 Argonaute and could act as its molecular chaperone. Crosses lacking HSP70-1 exhibit severe fertility defects, although growth and asexual sporulation are only mildly affected. In fission yeast, two Argonaute-binding (ARB) proteins mediate the loading of siRNAs onto Argonaute, which subsequently uses them to search for homologous mRNAs. The homologs in Neurospora, ARB1 and ARB2, also play a role in silencing. Deletion mutants of arb1 and arb2 act as semi-dominant suppressors of MSUD. While having a diffused localization in the cytoplasm, ARB1 and ARB2 preferentially accumulate outside of the nuclear envelope. The two ARB proteins interact with each other and with the SMS-2 Argonaute, supporting the notion that they help deliver siRNAs to the latter. In spite of their importance in sexual development, ARB1 and ARB2 do not appear to contribute to vegetative growth.
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Ph. D