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: HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing D-Glucose (4.5 g/L), L-Glutamine and 10% fetal bovine serum (FBS). Cells were seeded into 6-well plates and transiently transfected ~16 hours later, at ~80% confluency, using lipofectamine3000 (Invitrogen L3000001) CRISPR activaton experiments were transfected with either UniSAM empty vector (Addgene #99866) or UniSAM vector containing sgRNA targeting the promoter region of the targeted first exon. CRISPR inteference experiments were transfected with either dCas9-P2A-EGFP empty vector (Addgene #159086) or dCas9-P2A-EGFP vector containing sgRNA targeting downstream of the transcription start site of the targeted first exon. 48 hours post transfection, total RNA was extracted using RNeasy Mini Kit according to the manufacturer's instructions. Reverse transcription was performed in a reaction mix containing 1 µg of total RNA, using Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, K1652) according to the manufacturer's instructions. Two seperate batches with different experimental targets were sequenced. The first was sequenced xxxx. The second batch of Total RNA was sent to Novogene on dry ice. Novogene performed poly-A selection paired-end RNA-sequencing.Paired-end Illumina libraries were sequenced on Illumina Novaseq 6000.