Background: Gene regulation involves transcriptional and post-transcriptional proteins navigating the genome to locate precise DNA and RNA targets. Traditional models inadequately explain the rapid dynamics observed in nuclear regulatory processes. A new perspective suggests nuclear compartmentalization facilitates precise target localization, concentrating molecules necessary for compartment-specific gene regulation. Non-coding RNAs, such as Xist, form nuclear compartments through a seed-bind-recruit model, concentrating molecules like the transcriptional repressor Sharp and its partner HDAC3, essential for Xist-mediated X-inactivation. Recently, we found that protein-coding pre-mRNAs concentrate in transcriptional hubs, potentially seeding compartments for dynamic gene regulation. Sharp, a key regulator of compartment-based gene regulation, binds pre-mRNAs/mRNAs extensively, influencing their abundance. This suggests a non-coding role for pre-mRNAs/mRNAs in forming nuclear compartments and influencing gene expression.

Objective: To investigate the role of pre-mRNAs in forming nuclear compartments and controlling gene expression through direct interactions with Sharp.

Methods: Covalent-Linkage and Affinity Purification (CLAP) was used to purify RNAs bound directly to Sharp, revealing Sharp binding targets and sites. PHASTCONS analysis assessed the conservation of Sharp–pre-mRNA binding sites between mouse and human. An Auxin-degradable Sharp mESC line was used to study Sharp-pre-mRNA interactions in nuclear compartment formation and gene regulation. Gene expression changes were analyzed using qPCR, Western Blot, and Bulk and Single-Cell RNA sequencing. Sharp-pre-mRNA dependent nuclear compartmentalization was assessed using Conden-Sprite, a new SPRITE method revealing compartment-specific RNA-DNA contacts.

Results: CLAP data reveals that 95% of Sharp's pre-mRNA-binding targets are pre-mRNAs/mRNAs, with intronic binding sites conserved between mouse and human. Sharp depletion elevates transcription levels of target mRNAs, as shown by qPCR and RNA-Seq. Preliminary Conden-Sprite data indicates enrichment of RNA-DNA contacts between Sharp target pre-mRNAs and their corresponding gene loci.

Conclusions: These experiments suggest a new mechanism of gene dosage regulation where Sharp controls mRNA levels by binding pre-mRNAs and sensing active transcription levels. We hypothesize that pre-mRNAs seed dynamic Sharp-containing nuclear compartments near transcription sites, transiently silencing transcription via Sharp’s recruitment of HDAC3. Further experiments are required to fully elucidate this mechanism.