Global Analysis of the Human RNA Degradome Reveals Widespread Decapped and Endonucleolytic Cleaved Transcripts.

Jung-Im Won,Jeehee Yoon,Dong-Hoon Jeong,Jaemoon Shin,So Young Park

doi:10.3390/ijms21186452

Abstract

RNA decay is an important regulatory mechanism for gene expression at the posttranscriptional level. Although the main pathways and major enzymes that facilitate this process are well defined, global analysis of RNA turnover remains under-investigated. Recent advances in the application of next-generation sequencing technology enable its use in order to examine various RNA decay patterns at the genome-wide scale. In this study, we investigated human RNA decay patterns using parallel analysis of RNA end-sequencing (PARE-seq) data from XRN1-knockdown HeLa cell lines, followed by a comparison of steady state and degraded mRNA levels from RNA-seq and PARE-seq data, respectively. The results revealed 1103 and 1347 transcripts classified as stable and unstable candidates, respectively. Of the unstable candidates, we found that a subset of the replication-dependent histone transcripts was polyadenylated and rapidly degraded. Additionally, we identified 380 endonucleolytically cleaved candidates by analyzing the most abundant PARE sequence on a transcript. Of these, 41.4% of genes were classified as unstable genes, which implied that their endonucleolytic cleavage might affect their mRNA stability. Furthermore, we identified 1877 decapped candidates, including HSP90B1 and SWI5, having the most abundant PARE sequences at the 5′-end positions of the transcripts. These results provide a useful resource for further analysis of RNA decay patterns in human cells.

Highlights

The gene expression levels within a cell are determined by the interplay of tightly regulated processes for RNA synthesis and decay
Fragments per kilobase in million (FPKM) reads values from RNA-seq data were obtained for steady state mRNA levels (Figure 1A)
We compared the decay reads per kilobase in million (DPKM) and fragments per kilobase in million (FPKM) values of two biological replicates of Parallel analysis of RNA ends (PARE)-seq and RNA-seq data, respectively, in order to determine data reproducibility. This analysis revealed a high correlation in DPKM and FPKM values between the two biological replicates (RNA-seq R2 = 0.999; and PARE-seq R2 = 0.704), indicating the high quality and reproducibility of the data (Figure 1B)

Summary

Introduction

The gene expression levels within a cell are determined by the interplay of tightly regulated processes for RNA synthesis and decay. Cytoplasmic mRNAs are internally degraded by endoribonucleases or from one of the extremities by exoribonucleases These ribonucleolytic activities include decapping, 5 to 3 exonucleolytic decay, deadenylation, 3 to 5 exonucleolytic decay, and endonucleolytic cleavage [1,2,3,4,5]. The majority of cytoplasmic mRNA decay is initiated by a deadenylation-dependent pathway, which shortens the mRNA 3 poly(A) tail [2] This process is mediated by the activity of deadenylase complexes (i.e., CCR4–CAF1–NOT1) or PARN [2,6]. Endonucleolytic cleavage occurs within the body of the mRNA, followed by the degradation of the downstream fragment by XRN1 and the upstream fragment by the exosome Endoribonucleases, such as AGO, SMG6, and RRP44/DIS3, are involved in this pathway [9]. NMD targets that typically contain premature termination codons bypass deadenylation and undergo 5 cap removal by DCP2 and initiated by NMD factors, followed by 5 to 3 degradation by XRN1

Methods

Results

Discussion

Conclusion