IVT-SAPAS: Low-Input and Rapid Method for Sequencing Alternative Polyadenylation Sites.

Yu Xue,Yonggui Fu,Yutong Ge,Anlong Xu,Xiaojian Wu,Yu Sun,Liang Wan,Jiahui Liang

doi:10.1371/journal.pone.0145477

Abstract

Gene transcribing with alternative polyadenylation (APA) sites leads to mRNA isoforms, which may encode different proteins or harbor different 3'UTRs. APA plays an important role in regulating gene expression network among various physiological processes, such as development, immune responses and cancer. Several methods of library construction for APA study have been developed to apply high-throughput sequencing. However, the requirement of high-input RNA and time-consuming nature of the current methods limited the studies of APA for the samples difficult to obtain. Here, we describe a new method based on our SAPAS in combining in vitro transcription (IVT) and magnetic beads purification. The new IVT-SAPAS provides a rapid and high-parallel procedure for APA library construction with low-input sample, which may be a new robust approach for studying APA.

Highlights

Transcription termination by RNA Pol II (RNAPII) in eukaryotes is conducted with recognizing the poly(A) signals by cleavage and polyadenylation complex factors
Various biological effects associated with tandem alternative polyadenylation (APA) were investigated, including cancer transformation [1, 2], embryonic development [3,4,5] immune responses [6] and neuronal activity [7]
By integrating SAPAS, in vitro transcription with magnetic bead based purification and size selection, we developed IVT-SAPAS (Fig 1)

Summary

Introduction

Transcription termination by RNA Pol II (RNAPII) in eukaryotes is conducted with recognizing the poly(A) signals by cleavage and polyadenylation complex factors. Tandem APA sites located in 3'UTR region can lead to transcription of different mRNA isoforms with various 3'UTRs. Various biological effects associated with tandem APA were investigated, including cancer transformation [1, 2], embryonic development [3,4,5] immune responses [6] and neuronal activity [7]. Various biological effects associated with tandem APA were investigated, including cancer transformation [1, 2], embryonic development [3,4,5] immune responses [6] and neuronal activity [7] These studies found that shorter 3’UTRs may contribute to a higher proliferation rate of cells, cancer transformation, and responses to stimuli. One of the well known examples is the gene encoding μ chain of IgM

Methods

Results

Conclusion