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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 platform-dependent 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 lineage-specific 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.

SUBMITTER: Wu CS

PROVIDER: S-EPMC3875859 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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

Wu Chan-Shuo CS Yu Chun-Ying CY Chuang Ching-Yu CY Hsiao Michael M Kao Cheng-Fu CF Kuo Hung-Chih HC Chuang Trees-Juen TJ

Genome research 20131016 1

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 ...[more]

PMID: 24131564

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Project description:BACKGROUND: Transcriptional control of embryonic stem (ES) cell pluripotency has been a subject of intense study. Transcriptional regulators including Oct4 (Oct3/4 index), Sox2 and Nanog are fundamental for maintaining the undifferentiated state. However, the ES cell transcriptome is not limited to their targets, and exhibits considerable complexity when assayed with microarray, MPSS, cDNA/EST sequencing, and SAGE technologies. To identify novel genes associated with pluripotency, we globally searched for ES transcripts not corresponding to known genes, validated their sequences, determined their expression profiles, and employed RNAi to test their function. RESULTS: Gene Identification Signature (GIS) analysis, a SAGE derivative distinguished by paired 5' and 3' transcript end tags, identified 153 candidate novel transcriptional units (TUs) distinct from known genes in a mouse E14 ES mRNA library. We focused on 16 TUs free of artefacts and mapping discrepancies, five of which were validated by RTPCR product sequencing. Two of the TUs were revealed by annotation to represent novel protein-coding genes: a PRY-domain cluster member and a KRAB-domain zinc finger. The other three TUs represented intergenic splicing events involving adjacent, functionally unrelated protein-coding genes transcribed in the same orientation, with one event potentially encoding a fusion protein containing domains from both component genes (Clk2 and Scamp3). Expression profiling using embryonic samples and adult tissue panels confirmed that three of the TUs were unique to or most highly expressed in ES cells. Expression levels of all five TUs dropped dramatically during three distinct chemically induced differentiation treatments of ES cells in culture. However, siRNA knockdowns of the TUs did not alter mRNA levels of pluripotency or differentiation markers, and did not affect cell morphology. CONCLUSION: Transcriptome libraries retain considerable potential for novel gene discovery despite massive recent cDNA and EST sequencing efforts; cDNA and EST evidence for these ES cell TUs had been limited or absent. RTPCR and full-length sequencing remain essential in resolving the bottleneck between numerous candidate novel transcripts inferred from high-throughput sequencing and the small fraction that can be validated. RNAi results indicate that, despite their strong association with pluripotency, these five transcriptomic novelties may not be required for maintaining it.

Project description:Monitoring global gene expression provides insight into how genes and regulatory signals work together to guide embryo development. The fields of developmental biology and teratology are now confronted with the need for automated access to a reference library of gene-expression signatures that benchmark programmed (genetic) and adaptive (environmental) regulation of the embryonic transcriptome. Such a library must be constructed from highly-distributed microarray data. Birth Defects Systems Manager (BDSM), an open access knowledge management system, provides custom software to mine public microarray data focused on developmental health and disease. The present study describes tools for seamless data integration in the BDSM library (MetaSample, MetaChip, CIAeasy) using the QueryBDSM module. A field test of the prototype was run using published microarray data series derived from a variety of laboratories, experiments, microarray platforms, organ systems, and developmental stages. The datasets focused on several developing systems in the mouse embryo, including preimplantation stages, heart and nerve development, testis and ovary development, and craniofacial development. Using BDSM data integration tools, a gene-expression signature for 346 genes was resolved that accurately classified samples by organ system and developmental sequence. The module builds a potential for the BDSM approach to decipher a large number developmental processes through comparative bioinformatics analysis of embryological systems at-risk for specific defects, using multiple scenarios to define the range of probabilities leading from molecular phenotype to clinical phenotype. We conclude that an integrative analysis of global gene-expression of the developing embryo can form the foundation for constructing a reference library of signaling pathways and networks for normal and abnormal regulation of the embryonic transcriptome. These tools are available free of charge from the web-site http://systemsanalysis.louisville.edu requiring only a short registration process.

Dataset Information

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

Publications

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

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