Investigating the temporal recruitment of RNA-binding proteins to nascent intronless transcripts during mRNP formation

RNA-binding proteins (RBPs) are required for RNA maturation processes, including capping, splicing, polyadenylation, and export. However, our understanding of gene-specific sequential RBP recruitment for mRNP assembly has been limited. To investigate the temporal recruitment of RBPs to different regions of nascent transcripts at actively transcribing genes in real-time, we used fluorescently tagged RBPs and mRNAs in cell systems that allow the detection of actively transcribing genes. By employing an inducible, intronless long reporter gene, we mapped the sequential loading of factors onto active transcripts, independently of splicing. We measured a temporal gap between the early recruitment of 5′-end factors, such as CBP80, and the later arrival of 3′-end factors such as CPSF6, which correlates to an RNA polymerase II elongation rate of approximately 3.8 kb/min. In addition, we demonstrate the sequential recruitment of the m 6 A modification machinery, and observed that certain components of the TREX complex are robustly recruited, while exon-junction complex (EJC) subunits are notably absent from the active site of transcription of the intronless reporter genes. We find a hierarchical 3′-specific assembly, where the DEAD-box helicase DDX39B arrives prior to the export adaptor ALYREF, and the cotranscriptional recruitment of ALYREF to the nascent mRNA is critically dependent on the poly(A) binding protein PABP2, but not CPSF6. This study sheds light on the dynamic assembly of mRNPs, linking RBP recruitment to transcriptional control of gene expression.