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. Now, researchers from the Chinese Academy of Sciences have developed 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 they name poly(A) inclusive RNA isoform sequencing (PAIso-seq). Using this method, the researchers could quantify isoform specific poly(A) tail length. More interestingly, they found that 17% of the mRNAs harbor non-A residues within the body of poly(A) tails in mouse GV oocytes. The researchers 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.
The principle and validation of PAIso−seq
a Flowchart for PAIso−seq method design. The main steps of the method include end-extension, template-switching, full-length cDNA amplification, circular adaptor ligation, and PacBio sequencing. b The structure (top panel) and agarose gel analysis of the poly(A) spike-ins (middle panel). The mean poly(A) tail length of each spike-in measured by PAIso−seq (bottom panel). Source data are provided as a Source Data file. c The poly(A) tail lengths of Dnmt1, Btg4, and Plat in GV oocytes measured by PAIso−seq (density plot of poly(A) tail length of detected CCS reads of the given genes, three replicates, left panel) and PAT assay by using capillary electrophoresis on fragment analyzer (mean of three replicates, right panel). The mean length of each gene poly(A) tail measured by PAIso−seq is shown. The number of CCS reads used are 141 (Dnmt1/GV rep.1), 249 (Dnmt1/GV rep.2), and 165 (Dnmt1/SCGV com.); 164 (Btg4/GV rep.1), 521 (Btg4/GV rep.2), and 357 (Btg4/SCGV com.); 136 (Plat/GV rep.1), 277 (Plat/GV rep.2), and 207 (Plat/SCGV com.). The average length of poly(A) tails are 74 nt (Dnmt1), 44 nt (Btg4), and 45 nt (Plat) measured by the PAT assay. RFU, relative fluorescence units.