RBBP6 anchors pre-mRNA 3' end processing to nuclear speckles for efficient gene expression

Pre-mRNA 3' processing is an integral step in mRNA biogenesis. However, where this process occurs in the nucleus remains unknown. Here, we demonstrate that nuclear speckles (NSs), membraneless organelles enriched with splicing factors, are major sites for pre-mRNA 3' processing in human cells. We show that the essential pre-mRNA 3' processing factor RBBP6 associates strongly with NSs via its C-terminal intrinsically disordered region (IDR). Importantly, although the conserved N-terminal domain (NTD) of RBBP6 is sufficient for pre-mRNA 3' processing in vitro, its IDR-mediated association with NSs is required for efficient pre-mRNA 3' processing in cells. Through proximity labeling analyses, we provide evidence that pre-mRNA 3' processing for over 50% of genes occurs near NSs. We propose that NSs serve as hubs for RNA polymerase II transcription, pre-mRNA splicing, and 3' processing, thereby enhancing the efficiency and coordination of different gene expression steps. Overall design: To study the role of RBBP6 and its localization to nuclear speckles in pre-mRNA 3' processing, we conducted the following experiments: 1. Depleted RBBP6 and performed 4sU-seq to detect transcription readthrough. 2. Depleted RBBP6 and rescued it with either full-length or domain-deleted RBBP6 variants, then performed PAS-seq to detect changes in polyadenylation sites (PAS). 3. Overexpressed RBBP6 variants and performed PAS-seq. 4. Depleted nuclear speckle scaffold proteins and performed PAS-seq