Transcriptome-wide studies on eukaryotic mRNA tail

Abstract

Eukaryotic mRNA is subject to intensive post-transcriptional modifications, which critically influences mRNA stability and translatability. Newly synthesized mRNA acquires a 7-methlyguanosine cap at the 5′ end and polyadenosine tail at the 3′ end. In addition to such canonical modifications, recent studies have revealed untemplated nucleotide additions such as U-tail and G-tail, base modifications including N6-methyladenosine and pseudouridylation, and A-to-I editing, as epitranscriptomic signatures of mRNA. To investigate RNA tailing at the genomic scale, I recently developed a method called TAIL-seq which accurately measures poly(A) tail length and 3′ end modifications. Interestingly, I discovered that mammalian cells carried median 50–100 nt of poly(A) tail and widespread uridylation and guanylation at the downstream of poly(A) tail. Moreover, U-tails were mainly found on short poly(A) tails (<~25 nt), implicating its role in mRNA turnover. Uridylation has been observed on mRNAs in various species, yet its mechanism and significance remained unknown. By applying TAIL-seq, I identify TUT4 and TUT7 (TUT4/7), also known as ZCCHC11 and ZCCHC6, respectively, as mRNA uridylation enzymes in mammals. Uridylation readily occurs on deadenylated mRNAs in cells. Consistently, purified TUT4/7 selectively recognize and uridylate RNAs with short A tails (<~25 nt) in vitro. Moreover, PABPC1 antagonizes uridylation of polyadenylated mRNAs, contributing to the specificity for short A. In cells depleted of TUT4/7, the vast majority of mRNAs lose the oligo-U tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of oligo-uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 are required for enhanced decay of microRNA targets. My study explains the mechanism underlying selective uridylation of deadenylated mRNAs, and demonstrates a fundamental role of oligo-U-tail as a molecular mark for global mRNA decay. Next, I report a new version of TAIL-seq (mRNA TAIL-seq or mTAIL-seq), which increases sequencing depth for mRNAs by ~1,000 fold compared to the previous version. Original version of TAIL-seq provided a various information, but its low sensitivity precluded its application to minute amounts of biological materials. With the improved method, I investigate the regulation of poly(A) tail in Drosophila oocytes and embryos. I find that maternal mRNAs are polyadenylated mainly during late oogenesis, prior to fertilization, and further modulated upon egg activation. Wispy, a noncanonical poly(A) polymerase, adenylates most maternal mRNAs with a few intriguing exceptions such as ribosomal protein transcripts. By comparing mTAIL-seq data to ribosome profiling data, I further find a strong coupling between poly(A) tail length and translational efficiency during egg activation. My data suggest that regulation of poly(A) tail in oocytes shapes the translatomic landscape of embryos, thereby directing the onset of animal development. By virtue of the high sensitivity, low cost, technical robustness, and broad accessibility, mTAIL-seq will be a potent tool to improve our understanding of mRNA tailing. Taken together, I investigated mRNA tailings in eukaryotes by using sequencing methods that I developed, TAIL-seq and mTAIL-seq, and found their roles in the control of mRNA stability and translation.