SAF-A mutants disrupt chromatin structure through dominant negative effects on RNAs associated with chromatin

Abstract

Here we provide a brief review of relevant background before presenting results of our investigation into the interplay between scaffold attachment factor A (SAF-A), chromatin-associated RNAs, and DNA condensation. SAF-A, also termed heterogenous nuclear protein U (hnRNP U), is a ubiquitous nuclear scaffold protein that was implicated in XIST RNA localization to the inactive X-chromosome (Xi) but also reported to maintain open DNA packaging in euchromatin. Here we use several means to perturb SAF-A and examine potential impacts on the broad association of RNAs on euchromatin, and on chromatin compaction. SAF-A has an N-terminal DNA binding domain and C-terminal RNA binding domain, and a prominent model has been that the protein provides a single-molecule bridge between XIST RNA and chromatin. Here analysis of the impact of SAF-A on broad RNA-chromatin interactions indicate greater biological complexity. We focus on SAF-A's role with repeat-rich C₀T-1 hnRNA (repeat-rich heterogeneous nuclear RNA), shown recently to comprise mostly intronic sequences of pre-mRNAs and diverse long non-coding RNAs (lncRNAs). Our results show that SAF-A mutants cause dramatic changes to cytological chromatin condensation through dominant negative effects on C₀T-1 RNA's association with euchromatin, and likely other nuclear scaffold factors. In contrast, depletion of SAF-A by RNA interference (RNAi) had no discernible impact on C₀T-1 RNA, nor did it cause similarly marked chromatin changes as did three different SAF-A mutations. Overall results support the concept that repeat-rich, chromatin-associated RNAs interact with multiple RNA binding proteins (RBPs) in a complex dynamic meshwork that is integral to larger-scale chromatin architecture and collectively influences cytological-scale DNA condensation.