RNA Splicing Analysis: From In Vitro Testing to Single-Cell Imaging

Abstract

Techniques for analyzing RNA splicing variants are attracting significant interest. Significantly, splicing variability between individual cells is primarily responsible for gene expression heterogeneity. However, common techniques cannot directly detect splicing variants and are mainly based on in vitro static and ensemble analysis of total RNA extracted from a mass of cells, which omits spatial information and cell heterogeneity. Here, we review the current knowledge about the contribution of RNA splicing analysis techniques from cell-free to single cells. Enormous efforts aimed at elucidating the RNA splicing processes are expected to vastly enrich the fields of single-cell research, drug discovery, and clinical diagnostics. RNA splicing is a fundamental regulatory process of gene expression and plays a pivotal role in transcriptome complexity, cell-fate determination, and organism development. The detection of splicing products has attracted significant interest because aberrant splicing can lead to cell dysfunction and numerous diseases, such as cancer and neurodegeneration. Particularly, splicing variability between individual cells is primarily responsible for gene expression heterogeneity. Investigations into single-cell expression of RNA splicing variants offer a venue for resolving gene regulatory circuits, mapping intracellular localization, and classifying cell types. This review discusses the merits and technical challenges in transitioning RNA splicing detection from in vitro testing to single-cell analysis. In the end, we suggest some future directions for RNA splicing analysis and expect to inspire innovative works and discoveries in this field. RNA splicing is a fundamental regulatory process of gene expression and plays a pivotal role in transcriptome complexity, cell-fate determination, and organism development. The detection of splicing products has attracted significant interest because aberrant splicing can lead to cell dysfunction and numerous diseases, such as cancer and neurodegeneration. Particularly, splicing variability between individual cells is primarily responsible for gene expression heterogeneity. Investigations into single-cell expression of RNA splicing variants offer a venue for resolving gene regulatory circuits, mapping intracellular localization, and classifying cell types. This review discusses the merits and technical challenges in transitioning RNA splicing detection from in vitro testing to single-cell analysis. In the end, we suggest some future directions for RNA splicing analysis and expect to inspire innovative works and discoveries in this field. RNA splicing is the process of removing introns from pre-messenger RNA (pre-mRNA) and stitching exons together to yield a mature mRNA.1Maniatis T. Tasic B. 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During the second reaction, the free 5′ exon attacks the 3′ splice site (3′SS), generating a lariat intron and ligated exons (spliced mRNA) (Figure 1A).4Chen M. Manley J.L. Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches.Nat. Rev. Mol. Cell Biol. 2009; 10: 741-754Crossref PubMed Scopus (652) Google Scholar, 5Crotti L.B. Horowitz D.S. Exon sequences at the splice junctions affect splicing fidelity and alternative splicing.Proc. Natl. Acad. Sci. USA. 2009; 106: 18954-18959Crossref PubMed Scopus (0) Google Scholar One of the major discoveries in RNA splicing research is alternative splicing, which allows for the production of multiple mRNA splicing variants from a single gene. Alternative splicing patterns include exon skipping, alternative 5′SSs and 3′SSs, intron retention, and back splicing (Figure 1B). 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The RETF probe was first used for the detection of lariat RNA, an intermediate splicing product.39Furukawa K. Abe H. Tamura Y. Yoshimoto R. Yoshida M. Tsuneda S. Ito Y. Fluorescence detection of intron lariat RNA with reduction-triggered fluorescent probes.Angew. Chem. Int. Ed. 2011; 50: 12020-12023Crossref PubMed Scopus (22) Google Scholar Lariat RNA plays an important role in biological processes, such as causing gene silencing just like microRNA and protecting mRNA against degradation. Despite the biological importance of lariat RNA, the method for its detection is still limited.40Lambowitz A.M. Zimmerly S. Mobile group II introns.Annu. Rev. Genet. 2004; 38: 1-35Crossref PubMed Scopus (0) Google Scholar, 41Okamura K. Hagen J.W. Duan H. Tyler D.M. Lai E.C. The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila.Cell. 2007; 130: 89-100Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar, 42Ruby J.G. Jan C.H. Bartel D.P. 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Lett. 2012; 22: 7248-7251Crossref PubMed Scopus (6) Google Scholar These two probes are unable to react as a template on the pre-mRNA because of the far distance between two exons, but they can react as a template on the spliced mRNA since that intron is removed and two exons are ligated together after splicing (Figure 3B). This method has demonstrated the capability of drug screening and flexibility for its easy application to any genes by simply altering the sequence of probes. This method was the first reported on the detection of intron lariat RNA, which is superior to fluorescence resonance energy transfer (FRET) probes in its ability to discriminate lariat RNAs from pre-lariat RNAs. Although this method has great potential in real-time detection of lariat RNA or spliced mRNA in cells, there is still a need for signal amplification to improve the detection sensitivity. A grand challenge for precise quantification of RNA splicing variants is their sequence homology. The RNA splicing isoforms from a single gene always share the same sequence. For example, splicing isoforms Δ(11q, 333-) (the last 333 nt deleted from exon 11) and Δ(11q, 3642-) (the last 3,642 nt deleted from exon 11) from the breast cancer susceptibility gene 1 (BRCA1)44van ’t Veer L.J. Dai H. Van De Vijver M.J. He Y.D. Hart A.A. Mao M. Peterse H.L. van der Kooy K. Marton M.J. Witteveen A.T. et al.Gene expression profiling predicts clinical outcome of breast cancer.Nature. 2002; 415: 530-536Crossref PubMed Scopus (6804) Google Scholar all have the same exon sequences except for the last part of the sequence in exon 11. Thus, the unique exon-exon junction sequence is the key site to discriminate splicing isoforms. PCR-based methods, such as RT-PCR and real-time PCR, are the most common amplification strategies used to achieve highly sensitive detection of mRNA splice variants.45Yang D. Le J. 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High-throughput quantification of splicing isoforms.RNA. 2010; 16: 442-449Crossref PubMed Scopus (51) Google Scholar Ligating the two oligonucleotide probes at the splice junction sites is the most reliable and direct method for discriminating the splicing variants. To address the challenge, Li and coworkers48Wang H. Wang H. Duan X. Sun Y. Wang X. Li Z. Highly sensitive and multiplexed quantification of mRNA splice variants by direct ligation of DNA probes at exon junction and universal PCR amplification.Chem. Sci. 2017; 8: 3635-3640Crossref PubMed Google Scholar have developed a facile ligation-based method capable of detecting RNA splicing variants (Figure 4). In this assay, they employed T4 RNA ligase 2 to enhance the ligation specificity. Meanwhile, the 3′ end of one ligation probe was modified with two ribonucleotides to greatly increase the ligation efficiency.49Cheng Y. Zhang X. Li Z. Jiao X. Wang Y. Zhang Y. 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Moreover, the proposed approach can achieve multiplex detection of RNA splicing isoforms in one tube by using common gel electrophoretic separation and simply encoding the ligation probes with unique length corresponding to each RNA splicing variant. This assay has also been applied to total RNA extracted from different cell lines, showing its feasibility as a quantitative diagnostic tool. Given its superior sensitivity and excellent specificity, this approach will have wide applications in molecular diagnostics and biomedical research. Other than fluorescence methods, Raman scattering is the inelastic scattering of photons through chemical bonds that utilizes the characteristic vibrational energy in a specific molecular bond.51Wachsmann-Hogiu S. Weeks T. Huser T. Chemical analysis in vivo and in vitro by Raman spectroscopy–From single cells to humans.Curr. Opin. 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Deiters A. Conditional control of alternative splicing through light-triggered splice-switching oligonucleotides.J. Am. Chem. Soc. 2015; 137: 3656-3662Crossref PubMed Scopus (20) Google Scholar, 57Havens M.A. Duelli D.M. Hastings M.L. Targeting RNA splicing for disease therapy.Wiley Interdiscip. Rev. RNA. 2013; 4: 247-266Crossref PubMed Scopus (83) Google Scholar To date, how splicing control tools (such as antisense oligonucleotides [ASOs]) or a variety of other factors affect the multiple splicing processes still remains underexplored. This is mainly because it is difficult to simultaneously analyze the efficiency of each splicing step with high sensitivity. Currently available methods cannot differentiate the intermediate splicing products from the final splicing products and thus are not amenable to quantitatively evaluate the efficiency of a two-step splicing reaction (Table 1).Table 1Technique Information of In Vitro Methods for RNA Splicing AnalysisTypeEnzymeTarget ProductDetection EfficiencyPrincipleSpecialtyReferenceRETFnolariat intron, spliced mRNAmoderatefluorescence resonance energy transferdiscriminating lariat RNAs from pre-lariat RNAsFurukawa et al.39Furukawa K. Abe H. Tamura Y. Yoshimoto R. Yoshida M. Tsuneda S. Ito Y. Fluorescence detection of intron lariat RNA with reduction-triggered fluorescent probes.Angew. Chem. Int. Ed. 2011; 50: 12020-12023Crossref PubMed Scopus (22) Google Scholar and Tamura et al.43Tamura Y. Furukawa K. Yoshimoto R. Kawai Y. Yoshida M. Tsuneda S. Ito Y. Abe H. Detection of pre-mRNA splicing in vitro by an RNA-templated fluorogenic reaction.Bioorg. Med. Chem. Lett. 2012; 22: 7248-7251Crossref PubMed Scopus (6) Google ScholarLigation-based PCRyesalternative spliced mRNAhighligation, PCRhigh sensitivityWang et al.48Wang H. Wang H. Duan X. Sun Y. Wang X. Li Z. Highly sensitive and multiplexed quantification of mRNA splice variants by direct ligation of DNA probes at exon junction and universal PCR amplification.Chem. Sci. 2017; 8: 3635-3640Crossref PubMed Google ScholarRaman multiplexernoalternative spliced mRNAmoderatelocalize