(665b) Transcription of Proximal RNAs Can Regulate Gene Expression By Modulating Transcriptional Condensate Dynamics

Bio-molecular factories called transcriptional condensates facilitate the transcription of mRNAs in eukaryotic cells. These condensates are dense assemblies of proteins including transcription factors and transcriptional coactivators. They form through a phase separation mechanism at genomic regions having a high density of protein-binding DNA called super-enhancers (SE). Transcriptional condensates are important from a therapeutic standpoint as they control several genes associated with cell identity and cancer.

Many transcriptional proteins have positively charged disordered domains. Through these domains, they can interact with the negatively charged phosphate backbone of RNAs via screened electrostatic interactions and undergo complex coacervation. At low RNA concentrations, the RNA and protein condense together into a dense phase due to attractive interactions between the oppositely charged polymers. At high RNA concentrations, the dense phase dissolves due to a buildup of negatively charged RNA, which repel each other. The result is a re-entrant phase diagram at equilibrium where protein partitioning into the dense phase initially increases and then decreases upon titrating the RNA concentration.

However, living cells operate far away from equilibrium. Enhancer RNAs (eRNAs) are actively produced from SE regions and can interact with transcriptional proteins. Phase field equations based on a free energy can describe the transport of proteins due its interactions with other molecules. The rate of eRNA transcription depends on the local concentration of transcriptional proteins. We used phase field equations for proteins coupled with reaction diffusion equations for actively produced RNA species to model the spatiotemporal dynamics of concentrations of eRNAs and proteins. The transcription of mRNA happens over a slower time scale and the total amount of mRNA produced (gene expression) depends on the dynamics of protein concentration at the SE regions.

Transcriptional condensates reside in an environment where several other RNA species including long non-coding RNAs are also being actively transcribed. Prior studies show that active transcription of proximal RNAs can affect mRNA transcription from neighboring genes. However, the mechanism is not well understood. We hypothesize that localization and active transcription of proximal RNAs can regulate the dynamics of protein concentrations at the SE regions, which in turn affects mRNA transcription.

To investigate the consequences of this mechanism and make testable predictions, we modified the phase field + reaction diffusion equations described before to account for multiple RNA species. We investigated how the distance to the proximal RNA locus and its rate of transcription affects the dynamics of protein concentration at the SE region.

In the absence of active RNA transcription, we predict that proximal RNAs near SE regions accelerate recruitment of transcriptional proteins by electrostatic attraction when the local protein concentrations are small. Once enough protein accumulates to form a dense phase, some proximal RNA can also jump from its locus to the SE region due to the same electrostatic attraction and further speeds up protein recruitment. This jumping effect however does not happen if the RNA locus is too far away.

When there is active transcription, we predict that actively transcribing proximal RNAs can “accelerate” protein recruitment to the SE regions during early times in the dynamics due to favorable electrostatic interactions. If the transcription rates of the proximal RNAs are large, there is an accumulation of this RNA at later stages in the dynamics, which leads to unfavorable electrostatic repulsion between the RNA species and “shuts down” protein recruitment. The tussle between these two effects determines the dynamics of protein concentration at the SE region and the total amount of mRNA transcribed. Our model predicts that when transcription rate of the proximal RNAs is low, the “acceleration” effect dominates and they promote transcription from neighboring genes. On the other hand, quickly transcribing proximal RNAs repress transcription of neighboring genes.

In summary, we hypothesize that proximal RNAs can fine-tune mRNA transcription through its interactions with molecules in transcriptional condensates. This could be a novel mechanism by which proximal RNAs regulate expression of neighboring genes in cis, in a manner that depends only on their total charge and not their specific sequence or secondary structure. The consequences of this mechanism could explain several puzzles related to gene regulation by non-coding RNAs, such as (1) why non-coding RNAs promote gene expression in certain cases and repress gene expression in others (2) why the sequence of certain non-coding RNAs don’t affect their impact on gene expression and (3) why certain non-coding RNA loci are present at conserved genomic positions with respect to target genes.