Viruses and transposable DNA elements are examples of genetic parasites that can jump around and cause havoc to their host’s genome. Neurospora crassa, an orange bread mold, has an interconnected cellular network that makes it especially vulnerable to these parasites. Accordingly, this fungus maintains several genome surveillance systems to protect itself against potential threats. 

One of these systems is known as meiotic silencing by unpaired DNA (MSUD), which utilizes an RNA silencing machinery to prevent mobile DNA elements from becoming active in the course of sexual reproduction. During early meiosis, homologous chromosomes from each parent are paired together. If any DNA segment is missing a pairing partner, it is considered a potential intruder and is subject to meiotic silencing. Silencing begins when an unpaired gene is detected and an aberrant RNA (aRNA) is produced. This single-stranded RNA molecule is brought to the outer nuclear periphery (the perinuclear region of the cell), where a host of silencing proteins await. In successive steps, these proteins process the aRNA into short-interfering RNAs (siRNAs), which then use sequence homology to guide the Argonaute protein to find target mRNAs for destruction.

In this work, we characterized several silencing proteins that associate with the SMS-2 Argonaute, the effector protein of the MSUD pathway. SAD-9 is a DEAD-box RNA helicase that recruits the Argonaute to the perinuclear region, the surveillance checkpoint for RNAs that exit the nucleus. Heat shock protein 70 is a highly conserved protein found in virtually all organisms, and its cellular roles including protein folding and heat stress response. A Neurospora Hsp70 protein is important for silencing, possibly by folding the Argonaute into its open conformation to allow for siRNA loading. The siRNA loading step is carried out by two Argoanute binding proteins, ARB1 and ARB2. The proteins examined in this study interact and work closely with the Argonaute, and they play a role in both meiotic silencing and sexual sporulation. Overall, this work sheds light on the silencing pathway leading up to mRNA degradation in MSUD, i.e., how Argonaute is recruited and prepared to receive an incoming siRNA.

Doctoral Committee Members

• Dr. Patrick Shiu, Chair
• Dr. James Birchler
• Dr. Walter Gassmann
• Dr. Kathleen Newton

Publications

Sy VT, Boone EC, Xiao H, Vierling MM, Schmitz SF, Ung Q, Trawick SS, Hammond TM, Shiu PKT. A DEAD-box RNA helicase mediates meiotic silencing by unpaired DNA. G3 (Bethesda). 2023;13(8):jkad083. 

Xiao H, Vierling, MM, Kennedy, RF, Boone EC, Decker, LM, Sy VT, Haynes, JB, Williams, MA, Shiu, PKT. Involvement of RNA granule proteins in meiotic silencing by unpaired DNA. G3 (Bethesda). 2021;11(12):jkab326.

Boone EC, Xiao H, Vierling MM, Decker LM, Sy VT, Kennedy RF, Bonham MA, Schmitz SF, John AM, Hammond TM, et al. An NCBP3-domain protein mediates meiotic silencing by unpaired DNA. G3 (Bethesda). 2020;10(6):1919–1927.