In higher eukaryotes, multi-exon genes can encode different transcript isoforms via alternative splicing (AS), greatly increasing the diversity of proteins. A vast variety of isoforms generated by alternative splicing have specific roles in tissues or stages of development., Further, changes in normal isoform expression have phenotypic consequences and have been associated with disease . For example, alternative splicing of the BCL2L1 gene gives rise to two alternative transcripts that are highly similar but have antagonistic functions: The longer isoform, Bcl-XL, acts as an apoptotic inhibitor, while the shorter isoform, Bcl-XS, acts as an apoptotic activator (Figure 1) .
Figure 1. Schematic representation of alternative transcripts generated from the BCL2L1 gene locus. The large isoform, Bcl-XL, protects cells from programmed death, whereas the short form, Bcl-XS, promotes apoptosis.
Long non-coding RNAs (LncRNAs) can also be regulated through AS processing. For example, maternally expressed gene 3 (MEG3), a well-known LncRNA with tumor suppressor activity, exists in up to 12 distinct isoforms resulting from alternative splicing (Fig 2A, adapted from ). Although each of these RNA isoforms acts in a similar way to mediate p53-dependent tumor suppression, each exhibits a markedly different tissue-specific expression pattern (Fig. 2B and Table 1 ). These results suggest that the specific treatment regimen of cancer patients can be influenced by which RNA isoform of MEG3 each patient presents with.
Figure 2. Model of MEG3 RNA isoforms generated by AS. Common exons are in red. Exon 3a contains 40 extra nucleotides at the 3'-end of exon 3 (gold). From .
Similarly, the Dlx-5/6 ultraconserved region is transcribed to generate alternative noncoding RNA isoforms: Evf-1 and Evf-2. Evf-1 is a developmentally-regulated LncRNA, the function of which is still unclear. Evf-2 is the first LncRNA shown to be involved in organogenesis. Evf-2 specifically cooperates with Dlx-2 to increase the transcriptional activity of the Dlx-5/6 enhancer in a target- and homeodomain-specific manner. The Evf-2 LncRNA and the Dlx-2 protein form a stable complex in vivo, suggesting that the Evf-2 LncRNA activates transcriptional activity by directly influencing Dlx-2 activity .
Figure 3. Alternative processing of the Evf primary transcript. Alternative transcription initiation, exon splicing, and polyadenylation give rise to two different transcript isoforms, Evf-1 and Evf-2.
Even some individual genes can encode both protein-coding and noncoding isoforms under different conditions or developmental stages.  For example, the LncRNA TUC338 is transcribed from the uc.338 ultraconserved region. Although uc.338 is partially located within the PCBP2 gene on human chromosome 12 (Figure 4), TUC338 is expressed independently of PCBP2. Further, aberrant expression of TUC338 has been shown to promote the progression of hepatocellular carcinoma (HCC) cell growth .
Figure 4. Generation of an LncRNA by alternative splicing of a protein-coding mRNA. TUC338, an LncRNA derived from the ultraconserved region, is a product of alternative splicing of the larger poly(rC) binding protein 2 (PCBP2) mRNA. From .
Transcript-specific detection by the Arraystar Human Array V3.0
Traditional expression profiling array probes are designed to hybridize either singly or in clusters with the 3' end of the transcript. This 3'-bias cannot distinguish between overlapping transcripts. Conversely, the Arraystar Human LncRNA Array V3.0 uses a specific exon or splice junction probe, enabling the reliable and accurate detection of each individual transcript. These transcript-specific probes can even specifically detect transcripts that overlap with other transcripts on the sense strand, which are notoriously difficult to analyze (Figure 5).
Figure 5: Specificity Comparison between Our Probes and Probes of Other Companies
Using our specific exon and splice junction probes (Red lines), the Arraystar Human LncRNA Array V3.0 can identify two different transcripts of the BCL2L1 gene (BCL-XL and BCL-XS), which are produced by the same pre-mRNA and are functionally antagonistic. Traditional expression profiling arrays spotted with a 3'-bias probe (Green line, Company A), 3'-bias clustered long oligo probes (yellow lines, Company B) or 3'-bias clustered short oligo probes (violet lines, Company C) cannot distinguish between these splice variants.
Strategies for designing transcript-specific probes for the Arraystar Human Array V3.0
Each transcript sequence corresponding to the genome is divided into exon elements or splice junction elements based on the coordinates of these transcripts. Next, one probe is designed for each element. Finally, transcript-specific probes are selected, and are used to identify each isoform of the primary transcript.
Figure 6. Strategies for the design of transcript-specific probes.
The gene 1 primary transcript is alternatively processed to two mature transcripts: A and B. First, transcript sequences are divided into 4 exon elements (F1, F2, F3 and F4), and 3 splice junction elements (J1, J2 and J3). Next, each element is represented by a specific probe. Finally, transcript A-specific probes (F3, J3), and transcript B-specific probes (J2) are selected. F1, F2 and F4 can detect both transcripts, and so are used as gene-specific probes to measure gene 1 overall expression levels.
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Reference1. Kwan, T., et al., Genome-wide analysis of transcript isoform variation in humans. Nat Genet, 2008. 40(2): p. 225-31.
2. Boise, L.H., et al., bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell, 1993. 74(4): p. 597-608.
3. Zhou, Y., X. Zhang, and A. Klibanski, MEG3 noncoding RNA: a tumor suppressor. J Mol Endocrinol, 2012. 48(3): p. R45-53.
4. Zhang, X., et al., Maternally expressed gene 3 (MEG3) noncoding ribonucleic acid: isoform structure, expression, and functions. Endocrinology, 2010. 151(3): p. 939-47.
5. 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): p. 1470-84.
6. Orom, U.A., et al., Long noncoding RNAs with enhancer-like function in human cells. Cell, 2010. 143(1): p. 46-58.
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