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SE-lncRNA Research

Transcript-Specific Probes on the Super-Enhancer LncRNA Arrays

 

Introduction
In higher eukaryotic cells, multi-exon genes can produce transcript isoforms by alternative splicing, greatly increase the variations of the encoded proteins. Many alternatively spliced transcript isoforms play distinct roles in different tissues and developmental stages. The aberrant expression of these transcript isoforms can lead to phenotypical changes and associate with diseases [1].

Similarly, super-enhancer lncRNAs (SE-lncRNA) transcribed within the super-enhancer regions can also be alternatively spliced. For example, Dlx-5/6 super-enhancer region produces SE-lncRNA isoforms: Evf-1 and Evf-2 (Fig. 1). Evf-1 is a developmentally regulatory lncRNA. Its function is not yet clear. Evf-2 is the first discovered functional lncRNA in organogenesis. SE-lncRNA Evf-2 specifically interacts with Dlx-2 protein to stimulate the Dlx-5/6 enhancer activity. In vitro, Evf-2 binds Dlx-2 to form stable complex, suggesting its direct modulation of Dlx-2 to regulate the transcription activation [2].

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Figure 1. Dlx-5/6 super-enhancer region produces two SE-lncRNA transcript isoforms Evf-1 and Evf-2 by alternative splicing.

Colorectal cancer specific CCAT1 is a SE-lncRNA transcribed in the super-enhancer region upstream of the MYC oncogene. CCAT1 has two transcript isoforms: CCAT1-L and CCAT1-S. CCAT1-L entirely overlaps with CCAT1-S and extends 2600-bp further at the 3’-end (Fig. 2). The expression levels for CCAT1-L and CCAT1-S are both increased in colorectal cancer tissues, with CCAT1-L far more than CCAT1-S. CCAT1-L is the one having the enhancer function at its transcription site to form chromatin loops with the MYC promoter, thus regulating the MYC gene expression [3].

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Figure 2. Two SE-lncRNA isoforms CCAT1-L and CCAT1-S are produced from the MYC super-enhancer region. Arraystar SE-LncRNA Array Probe A (red bar) specifically detects CCAT1-L without cross-hybridization with CCAT1-S.

SE-lncRNA transcript-specific detection on Arraystar Super-enhancer LncRNA Arrays
As alternatively spliced transcript isoforms are functionally significant, Arraystar Super-enhancer LncRNA Arrays are designed to reliably and accurately profile the isoforms by the array probes targeting specifically to the splice junctions or exon sequences only unique to each isoform (Fig. 2). These probes are even able to detect otherwise difficult transcripts that overlap with the sense-strand transcripts.
Arraystar SE-lncRNA Array transcript-specific probe design strategy is to first define the exons and splice junctions for each transcript as the elements. Probes are then designed for each such element and one best transcript-specific probe that can unambiguously distinguish all the transcript isoforms is qualified and selected (Fig. 3).

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Figure 3. Transcript-specific probe design strategy. The primary transcript of Gene1 is alternatively spliced into two mature transcripts A and B. These transcripts have a total of 4 unique exons (F1, F2, F3, F4) and 3 splice junctions (J1, J2, J3) as the “elements”. Each probe is then designed for each element. Finally, J2 is selected specifically for transcript A; F3 and J3 are selected specifically for transcript B. Probes for F1, F2 and F4 are common for transcripts A and B, which can be used as gene-specific probes for the overall level of Gene1 expression.

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References
1. Kwan T et al: Genome-wide analysis of transcript isoform variation in humans. Nat Genet 2008, 40(2):225-231.[PMID: 18193047]
2. Feng J et al: The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator. Genes Dev 2006, 20(11):1470-1484.[PMID: 16705037]
3. Xiang JF et al: Human colorectal cancer-specific CCAT1-L lncRNA regulates long-range chromatin interactions at the MYC locus. Cell Res 2014, 24(5):513-531.[PMID: 24662484]

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