mRNA Initiation and Termination are Spatially Coordinated

ABSTRACT: The expression of a precise mRNA transcriptome is crucial for establishing cell identity andfunction, with dozens of alternative isoforms produced for a single gene sequence. The regulationof mRNA isoform usage occurs by the coordination of co-transcriptional mRNA processingmechanisms across a gene. Decisions involved in mRNA initiation and termination underlie thelargest extent of mRNA isoform diversity, but little is known about any relationships betweendecisions at both ends of mRNA molecules. Here, we systematically profile the joint usage ofmRNA transcription start sites (TSSs) and polyadenylation sites (PASs) across tissues andspecies. Using both short and long read RNA-seq data, we observe that mRNAs preferentiallyusing upstream TSSs also tend to use upstream PASs, and congruently, the usage ofdownstream sites is similarly paired. This observation suggests that mRNA 5' end choice maydirectly influence mRNA 3' ends. Our results suggest a novel “Positional Initiation-TerminationAxis” (PITA), in which the usage of alternative terminal sites are coupled based on the order inwhich they appear in the genome.To examine the direct influence of promoter (AFE) choice on ALE decisions, we performed a series of CRISPR modulations on HEK293 cells to either activate or repress AFEs and measure the change in ALE usage. Overall design: K562 cells passage 10-12 were grown in RMPI medium supplemented with cells were treated with DRB 100uM for 3 hours. During the last 5 minnutes of DRB, 4su was added to a final concentration of 1mM. After PBS wash, cells were resuspended in 4su 1mM for a 10m labeling. Treated cells were transferred to low-bind tubes, spin down 2 minutes at 1000rcf, followed by Trizol/Chloroform extraction. Then, the aqueous phase was cleanup using the zymo-clean up kit. We enriched labeled RNA using MTSEA biotin-XX pulldown, followed by RNAseH-based ribosomal RNA depletion. Then, we did a polyI tailing and direct RNA nanopore library prep. For cDNA libraries, polyI tailing was followed by strand-switching RT and PCR, followed by cdna library prep following manufacture's instructions.