Abstract
Memory formation requires coordinated control of gene expression, protein synthesis, and ubiquitin-proteasome system (UPS)-mediated protein degradation. The catalytic component of the UPS, the 26S proteasome, contains a 20S catalytic core surrounded by two 19S regulatory caps, and phosphorylation of the 19S cap regulatory subunit RPT6 at serine 120 (pRPT6-S120) has been widely implicated in controlling activity-dependent increases in proteasome activity. Recently, RPT6 was also shown to act outside the proteasome where it has a transcription factor-like role in the hippocampus during memory formation. However, little is known about the proteasome-independent function of "free" RPT6 in the brain or during memory formation and whether phosphorylation of S120 is required for this transcriptional control function. Here, we used RNA-sequencing along with novel genetic approaches and biochemical, molecular, and behavioral assays to test the hypothesis that pRPT6-S120 functions independently of the proteasome to bind DNA and regulate gene expression during memory formation. RNA-sequencing following siRNA-mediated knockdown of free RPT6 revealed 46 gene targets in the dorsal hippocampus of male rats following fear conditioning, where RPT6 was involved in transcriptional activation and repression. Through CRISPR-dCas9-mediated artificial placement of RPT6 at a target gene, we found that RPT6 DNA binding alone may be important for altering gene expression following learning. Further, CRISPR-dCas13-mediated conversion of S120 to glycine on RPT6 revealed that phosphorylation at S120 is necessary for RPT6 to bind DNA and properly regulate transcription during memory formation. Together, we reveal a novel function for phosphorylation of RPT6 in controlling gene transcription during memory formation.
Keywords:
CRISPR; RPT6; hippocampus; memory; proteasome; transcription.