Poly(A) inclusive RNA isoform sequencing (PAIso−seq) reveals wide-spread non-adenosine residues within RNA poly(A) tails

Abstract

Message RNA poly(A) tails are vital for their function and regulation. However, the full-length sequence of mRNA isoforms with their poly(A) tails remains undetermined. Here, we develop a method at single-cell level sensitivity that enables quantification of poly(A) tails along with the full-length cDNA while reading non-adenosine residues within poly(A) tails precisely, which we name poly(A) inclusive RNA isoform sequencing (PAIso−seq). Using this method, we can quantify isoform specific poly(A) tail length. More interestingly, we find that 17% of the mRNAs harbor non-A residues within the body of poly(A) tails in mouse GV oocytes. We show that PAIso−seq is sensitive enough to analyze single GV oocytes. These findings will not only provide an accurate and sensitive tool in studying poly(A) tails, but also open a door for the function and regulation of non-adenosine modifications within the body of poly(A) tails.

Immunoglobulin expression and the humoral immune response is regulated by the non-canonical poly(A) polymerase TENT5C

“…This experimental strategy offers high-quality, full-length mRNA sequences, including UTRs and poly(A) tails giving deeper insights into transcriptome shaping, comparing to classical RNAseq experiments 32 . It is characterized by a relative technical simplicity, and in contrast to other RNA-seq methods, no PCR biases are introduced into libraries as in case of other currently used RNA 3ʹ-end research techniques, such as Nascent RNAend-Seq, TAIL-seq, PAL-seq, TED-seq, PAC-Seq, EnD-seq, FLAM-seq, or most recently PAIso-seq 31,[35][36][37][38][39][40][41] . Importantly, despite the recent dynamic expansion of RNA 3ʹ-terminome research, little was known how cytoplasmic ncPAP enzymes contribute to gene expression programs since such techniques were never applied for KOs of individual enzymes in physiological conditions.…”

So close, no matter how far: multiple paths connecting transcription to mRNA translation in eukaryotes

“…(iv) PAIso (poly(A) inclusive RNA isoform)-seq: Similar to FLAM-seq, it employs the PacBio platform to enable full-length sequencing of RNA. The 3 0 PCR handles are added by Klenow enzyme, and subsequent reverse transcription with 5 0 template switching enables the creation of circular cDNA libraries (Liu et al, 2019b). (v) PAT (poly(A) test RNA)-seq: RNA fragments bearing poly(A) tails are isolated and used for library construction (Harrison et al, 2015).…”

miRNA-dependent poly(A) length control in uncoupling transcription and translation of haploid male germ cells

“…The poly(A) length has long been known to regulate transcript stability and translational efficiency [2,3]. However, investigations into the role of poly(A) length control have just started to emerge [2,8,34,57]. This is primarily due to a lack of sensitive methodologies that allow for accurate determination of the full-length poly(A) tail sequences.…”