An RNA-Seq Strategy to Detect the Complete Coding and Non-Coding Transcriptome Including Full-Length Imprinted Macro ncRNAs

Abstract

Imprinted macro non-protein-coding (nc) RNAs are cis-repressor transcripts that silence multiple genes in at least three imprinted gene clusters in the mouse genome. Similar macro or long ncRNAs are abundant in the mammalian genome. Here we present the full coding and non-coding transcriptome of two mouse tissues: differentiated ES cells and fetal head using an optimized RNA-Seq strategy. The data produced is highly reproducible in different sequencing locations and is able to detect the full length of imprinted macro ncRNAs such as Airn and Kcnq1ot1, whose length ranges between 80–118 kb. Transcripts show a more uniform read coverage when RNA is fragmented with RNA hydrolysis compared with cDNA fragmentation by shearing. Irrespective of the fragmentation method, all coding and non-coding transcripts longer than 8 kb show a gradual loss of sequencing tags towards the 3′ end. Comparisons to published RNA-Seq datasets show that the strategy presented here is more efficient in detecting known functional imprinted macro ncRNAs and also indicate that standardization of RNA preparation protocols would increase the comparability of the transcriptome between different RNA-Seq datasets.

Macro lncRNAs

“…Imprinted lncRNAs have been identified at several imprinted gene clusters and are unusual as they show inefficient co-transcriptional splicing and polyadenylation and have been termed "macro" lncRNAs to reflect that their main product is unspliced, in contrast to the fully-spliced Xist lncRNA and all known lincRNAs. 21,29,30 In this review, we focus on functional cis-acting macro lncRNAs and their role in disease and review three main areas: (1) imprinted cis-acting macro lncRNAs, their unusual transcript biology and how they differ from spliced lncRNAs, (2) non-imprinted cis-acting macro lncRNAs linked to disease and (3), methods available to test the functions of large data sets of newly annotated but still uncharacterized lncRNAs.…”

“…The Airn and Kcnq1ot1 macro lncRNAs are also distinguished by a relatively short half-life, a high interspersed repeat content and low sequence conservation. 51,60,70 One additional feature visible from genome-tiling-array and RNA-seq analysis 30,60,71 is that the Airn and Kcnq1ot1 macro lncRNAs show reduced RNA abundance toward the 3' end of the gene, indicating imprecise termination. Together these features indicate that the macro lncRNA product may be less important than its transcription in regulating imprinted expression.…”

“…Therefore, an effective RNA-seq strategy to discover novel macro lncRNAs should include an efficient rRNA removal step to allow an unbiased view of the complete nonribosomal transcriptome. 30 In addition, strand-specific RNA-seq is necessary to identify overlapping lncRNAs (e.g., Airn) and that overlap silenced genes, may induce a form of transcriptional interference either of the promoter or of essential enhancers. 41,72,73 These models do not exclude that the macro lncRNA product may also have a functional role for example, to repress non-overlapping genes.…”

“…Some studies have shown that macro lncRNAs such as Airn and Kcnq1ot1 are better visible in RNA-seq data sets produced by a pipeline that replaces polyA + selection with rRNA depletion. 30,69 One unsolved problem is how to determine if ultra-long macro lncRNAs are one continuous transcript or composed of transcripts from clusters of smaller genes. For the Airn and Kcnq1ot1 macro lncRNAs continuous transcription was demonstrated by laborious experiments that demonstrated their repressor function, 35,36 since insertion of a polyA signal downstream of the promoter removed all downstream transcripts.…”

“…Since lncRNAs are also generally lowly expressed relative to protein-coding genes, 21,30 it is also not known if some could arise from "opportunistic" non-specific RNA polymerase activity that reflects an open chromatin state (see discussion in ref. 66).…”