Extension of human lncRNA transcripts by RACE coupled with long-read high-throughput sequencing (RACE-Seq)

Long non-coding RNAs (lncRNAs) constitute a large, yet mostly uncharacterized fraction of the mammalian transcriptome. Such characterization requires a comprehensive, high-quality annotation of their gene structure and boundaries, which is currently lacking. Here we describe RACE-Seq, an experimental workflow designed to address this based on RACE (rapid amplification of cDNA ends) and long-read RNA sequencing. We apply RACE-Seq to 398 human lncRNA genes in seven tissues, leading to the discovery of 2,556 on-target, novel transcripts. About 60% of the targeted loci are extended in either 5′ or 3′, often reaching genomic hallmarks of gene boundaries. Analysis of the novel transcripts suggests that lncRNAs are as long, have as many exons and undergo as much alternative splicing as protein-coding genes, contrary to current assumptions. Overall, we show that RACE-Seq is an effective tool to annotate an organism’s deep transcriptome, and compares favourably to other targeted sequencing techniques

Whole-transcriptome analysis of UUO mouse model of renal fibrosis reveals new molecular players in kidney diseases

Transcriptome analysis by RNA-seq technology allows novel insights into gene expression and regulatory networks in health and disease. To better understand the molecular basis of renal fibrosis, we performed RNA-seq analysis in the Unilateral Ureteric Obstruction (UUO) mouse model. We analysed sham operated, 2- and 8-day post-ligation renal tissues. Thousands of genes with statistical significant changes in their expression were identified and classified into cellular processes and molecular pathways. Many novel protein-coding genes were identified, including critical transcription factors with important regulatory roles in other tissues and diseases. Emphasis was placed on long non-coding RNAs (lncRNAs), a class of molecular regulators of multiple and diverse cellular functions. Selected lncRNA genes were further studied and their transcriptional activity was confirmed. For three of them, their transcripts were also examined in other mouse models of nephropathies and their up- or down-regulation was found similar to the UUO model. In vitro experiments confirmed that one selected lncRNA is independent of TGFβ or IL1b stimulation but can influence the expression of fibrosis-related proteins and the cellular phenotype. These data provide new information about the involvement of protein-coding and lncRNA genes in nephropathies, which can become novel diagnostic and therapeutic targets in the near future