Molecular dissection of the roles of RNA processing proteins in meiotic silencing by unpaired DNA
Authors:   , Vierling Michael M. 
Published: September 2022

[EMBARGOED UNTIL 6/1/2023] The filamentous fungus Neurospora crassa is a model organism for the study of genome defense and epigenetic mechanisms. N. crassa utilizes meiotic silencing by unpaired DNA (MSUD) to silence unpaired genes during sexual development. MSUD requires two steps: a sequence is identified as unpaired during meiosis and any RNA homologous to that unpaired sequence is targeted for degradation. The mechanism accomplishing this degradation is RNA interference (RNAi), and many genes important to this pathway have been identified. Once a signal is produced from an unpaired sequence, most likely an RNA molecule, that RNA is converted into small interfering RNAs (siRNAs) that act as sequence specific guides for mRNA destruction. MSUD is presumed to be a strong deterrent of transposons and viruses, which can cause detrimental mutations as they proliferate and insert themselves randomly into the host's genome. The cap-binding complex (CBC) is composed of cap-binding protein 80 (CBP80) and cap-binding protein 20 (CBP20), which bind the 7-methylguanosine cap of mRNA. The CBC has been implicated in being important for steps of mRNA maturation such as splicing and for aiding mRNA export from the nucleus. Work from multiple model organisms has implicated the CBC in small RNA-mediated post transcriptional silencing of mRNA, and in Neurospora, removal of either or both CBC components has been shown to reduce the efficacy of MSUD. The CBC may mediate MSUD through its interaction with the Argonaute protein suppressor of meiotic silencing-2 (SMS-2) by delivering mRNA to SMS-2 for silencing. Elimination of either CBC component causes a reduction in sexual development but does not hamper vegetative growth. Another MSUD effector protein called SAD-8 has homology to a recently discovered mammalian protein, nuclear cap-binding protein 3 (NCBP3). Crosses in a sad-8-null background are almost completely deficient in MSUD activity, indicating that SAD-8 is a crucial player of MSUD. SAD-8 interacts with CBP20 and CBP80 (likely forming a trimer) and has a mostly nuclear localization. Also, SAD-8 and SMS-2 interact in the perinuclear region of spore sacs, which further supports a connection between the CBC and MSUD silencing complex (MSC). SAD-8, unlike many other MSUD proteins, is not important for sexual sporulation. P-bodies are cytoplasmic foci found in eukaryotic cells. They are implicated not only in normal turnover of mRNAs, but also targeted translational repression and destruction of specific mRNA. Two proteins that localize to P-bodies, CGH-1 and CAR-1, have been identified through our work as MSUD mediators. These proteins reside in both P-bodies and the perinuclear region of Neurospora spore sacs. CAR-1 and CGH-1 only interact directly in P-bodies, but they interact with several MSUD proteins (including SMS-2) in the perinuclear region. Both proteins are involved in vegetative growth and sexual development as both phases of the life cycle are affected by their removal. ARS2 is a direct binding partner of the CBC in many organisms and is theorized to be a sorting platform for diverse co-transcriptional complexes being recruited to the CBC. One such cofactor believed to be recruited to the CBC is NCBP3 in human cells. NCBP3's ortholog in Neurospora crassa, SAD-8, is a known MSUD mediator and previous results showed that ARS2 also mediates MSUD. Upon further investigation, ARS2 was shown to interact with both SAD-8 and CBC, supporting the notion that ARS2 helps recruit the former to the latter. ARS2 also interacts with SMS-2, supporting the idea that the CBC/ARS2/SAD-8 complex can help SMS-2 to more efficiently capture exported mRNAs. Besides silencing, ARS-2 also plays a role in linear growth and ascospore production.