Elucidating the coordination of RNA processing using short-read and long-read RNA-sequencing methods

The maturation of mRNAs is crucial for gene regulation and proteome diversification. Transcripts are processed co-transcriptionally through a complex interplay of mechanisms that involve numerous protein machineries. In eukaryotes, most genes undergo alternative RNA processing through the context-dependent use of transcription start sites (TSSs), splice sites and polyadenylation sites. The accurate measurement of alternative TSS usage, alternative splicing and alternative polyadenylation has been enabled by short-read RNA-sequencing technologies. However, elucidating the timing, coordination and functional outcomes of alternative RNA processing is challenging, especially in vivo. The development of long-read sequencing (LRS) methodologies enables the characterization of various aspects of co-transcriptional RNA processing, each methodology providing unique perspectives and limitations. In this Review, we discuss recent advances in short-read sequencing and LRS technologies that measure transcripts in their nascent and mature state and at single-cell resolution and with whole-molecule read length in the case of LRS. We integrate new findings that functionally link alternative TSS, alternative splicing and alternative polyadenylation, with new implications for diseases such as cancer and neurodevelopmental and neurodegenerative disorders. Finally, we discuss insights gained using CRISPR tools into the coordination of RNA processing events.