Analysis of Transcriptome Complexity Through RNA Sequencing in Normal and Failing Murine Hearts

Abstract

Rationale Accurate and comprehensive de novo transcriptome profiling in heart is a central issue to better understand cardiac physiology and diseases. Although significant progress has been made in genome-wide profiling for quantitative changes in cardiac gene expression, current knowledge offers limited insights to the total complexity in cardiac transcriptome at individual exon level. Objective To develop more robust bioinformatic approaches to analyze high-throughput RNA sequencing (RNA-Seq) data, with the focus on the investigation of transcriptome complexity at individual exon and transcript levels. Methods and Results In addition to overall gene expression analysis, the methods developed in this study were used to analyze RNA-Seq data with respect to individual transcript isoforms, novel spliced exons, novel alternative terminal exons, novel transcript clusters (i.e., novel genes) and long non-coding RNA genes. We applied these approaches to RNA-Seq data obtained from mouse hearts following pressure-overload induced by trans-aortic constriction. Based on experimental validations, analyses of the features of the identified exons/transcripts, and expression analyses including previously published RNASeq data, we demonstrate that the methods are highly effective in detecting and quantifying individual exons and transcripts. Novel insights inferred from the examined aspects of the cardiac transcriptome open ways to further experimental investigations. Conclusions Our work provided a comprehensive set of methods to analyze mouse cardiac transcriptome complexity at individual exon and transcript levels. Applications of the methods may infer important new insights to gene regulation in normal and disease hearts in terms of exon utilization and potential involvement of novel components of cardiac transcriptome.

RNA Splicing

“…72 In hypertrophied and failing mouse hearts, alternative splicing profiles are likewise altered, with the largest differences in failing hearts. 73 In humans, Kong et al 74 were the first to use a genome-wide approach to study alternative splicing changes in the diseased heart. The splicing profile of diseased and control hearts differed extensively, and subsequently, the authors were able to correctly assign samples to control or disease based solely on the splicing profile.…”

Interplay of chromatin modifications and non-coding RNAs in the heart

“…RNA sequencing of the myocardium has revealed specific transcriptome profiles for coding and noncoding transcripts that distinguish the stages of the failing heart. 65 Recent studies have identified more than 1300 previously unannotated exons with altered expression levels in animal models of heart failure. 65 Among these, almost 682 exons displayed Figure 1.…”

“…65 Recent studies have identified more than 1300 previously unannotated exons with altered expression levels in animal models of heart failure. 65 Among these, almost 682 exons displayed Figure 1. interplay of chromatin modifications and non-coding RNAs regulate MHC genes in the heart.…”

Global impact of RNA splicing on transcriptome remodeling in the heart

“…Therefore, to generate a comprehensive expression profile for each exon, it is essential to perform the RNA-seq at sufficient depth. One major advantage of RNA-seq over micro-arrays is the possibility to identify novel, un-annotated exons or transcripts (Daines et al, 2011;Lee et al, 2011;Concha et al, 2012). In a recent study, we have developed two bioinformatic tools, guided transcriptome reconstruction and de novo reconstruction.…”

“…In a recent study, we have developed two bioinformatic tools, guided transcriptome reconstruction and de novo reconstruction. These tools allow detection of novel exons in known genes and novel transcript clusters (NTCs) (Lee et al, 2011).…”