Integrative transcriptome sequencing identifies trans-splicing events with important roles in human embryonic stem cell pluripotency

Abstract

Trans-splicing is a post-transcriptional event that joins exons from separate pre-mRNAs. Detection of trans-splicing is usually severely hampered by experimental artifacts and genetic rearrangements. Here, we develop a new computational pipeline, TSscan, which integrates different types of high-throughput long-/short-read transcriptome sequencing of different human embryonic stem cell (hESC) lines to effectively minimize false positives while detecting trans-splicing. Combining TSscan screening with multiple experimental validation steps revealed that most chimeric RNA products were platformdependent experimental artifacts of RNA sequencing. We successfully identified and confirmed four trans-spliced RNAs, including the first reported trans-spliced large intergenic noncoding RNA (''tsRMST ''). We showed that these trans-spliced RNAs were all highly expressed in human pluripotent stem cells and differentially expressed during hESC differentiation. Our results further indicated that tsRMST can contribute to pluripotency maintenance of hESCs by suppressing lineagespecific gene expression through the recruitment of NANOG and the PRC2 complex factor, SUZ12. Taken together, our findings provide important insights into the role of trans-splicing in pluripotency maintenance of hESCs and help to facilitate future studies into trans-splicing, opening up this important but understudied class of post-transcriptional events for comprehensive characterization.

Is an observed non-co-linear RNA product spliced intrans, incisor justin vitro?

“…This is consistent with the earlier reports that intragenic splicing is more common than intergenic splicing, and that the majority of observed intergenic trans -splicing candidates are, in fact, experimental artifacts (9,27). A possible explanation is that intragenic splicing within a single gene results in a higher local concentration of transcripts than intergenic splicing between different genes.…”

“…Primate ESC colonies were manually expanded to new feeder cells every four days (9,45). Mouse ESC D3 and K1 derived from C57BL/6 × ICR embryos were cultured in DMEM containing 15% FBS (level), 1x NEAA, 2 mM L-glutamine (Invitrogen) and 103 units/ml of leukemia inhibitory factor (Millipore) on Mitomycin C-treated MEF feeders (5 × 10 4 cells/cm 2 ).…”

Circular RNA enrichment in platelets is a signature of transcriptome degradation

“…These diverse lines of evidence suggest that there may not be a straightforward relationship between RT specificity and artifacts as previously suggested. 43 Finally, cellular levels of circRNAs are known to be low in proliferating and neoplastic human cells 44,60 but to increase during differentiation of ES cells 26 and during Drosophila development, 61 suggesting that they may be a biomarker for aging. Among nucleated tissues, they are highest in both human and Drosophila neural tissues 44,61 and are particularly enriched in synapses.…”

“…9,11,15,17,18 Transcripts with rearranged exon order are now known to be common in eukaryotic organisms and are characterized by backsplice exon junctions inconsistent with the underlying genomic DNA. [19][20][21][22][23][24] Evidence for linear and polyadenylated structures has been presented, 20,25,26 but it is now clear that most are circular (circRNAs) and cytoplasmic. [21][22][23] Recent experiments have established that intronic repeats flanking rearranged exons can promote circularization and that circRNAs primarily use canonical splice sites [27][28][29][30] and can be regulated by RNA binding proteins.…”

“…[21][22][23] Recent experiments have established that intronic repeats flanking rearranged exons can promote circularization and that circRNAs primarily use canonical splice sites [27][28][29][30] and can be regulated by RNA binding proteins. 29,31 Although 2 noncoding circRNAs act as miRNA sponges, 22,26 and circRNAs with retained introns (exon-intron or EIcircRNAs) can enhance transcription of their parental genes through interaction with U1 snRNP and RNA PolII, 32 the vast majority of circRNAs consist of exons from protein encoding genes and have no known function.…”