RNA Splicing Harnessing Introns to Monitor Gene Expression

Abstract

A number of new technologies have utilized synthetic RNAs which leverage the cell’s RNA splicing machinery to drive expression of gene products. Now a new study reports a technique to dynamically and non-invasively monitor gene expression by embedding reporters within introns contained in the parent gene.

Monitoring expression of a gene from its endogenous locus and promoter with reporter constructs typically requires disruption of protein-coding sequence or risk of altering gene expression. For example, replacement of a gene’s protein coding sequences with sequences encoding fluorescent or luminescent proteins provides a means to monitor native gene expression but ablates the endogenous gene sequence¹. Attempts to avoid tampering with protein coding sequences by inserting 2A skipping peptides can leave scars on the resulting chimeric protein.² Inserting reporter sequences in the 3’ untranslated region (3’UTR) of a gene can circumvent alterations in protein sequence but run the risk of tampering within endogenous mRNA expression, stability, and localization regulated by the 3’ UTR.³ Furthermore, these methods cannot be used to monitor expression of non-protein coding genes such as long non-coding RNAs (lncRNAs). More recently, CRISPR–Cas-based detection methods have been developed to identify and monitor RNA species, but these methods require linking of guide RNAs to a gene along with expression of CRISPR-Cas enzymes within cells⁴ which is cumbersome for in vivo studies. Given the above challenges, the most commonly utilized methods for evaluating gene expression are based on RT-PCR, RNA-seq, and RNA FISH, which often are limited to evaluation of gene expression from a single timepoint due to cost and labor-intensity⁵. Now, a new study reported in this issue of Nature Cell Biology reports a clever new technique to dynamically and non-invasively monitor gene expression by embedding reporters within artificial introns inserted into the parent gene⁶.

To circumvent the perturbation in nucleotide and protein sequence potentially generated by conventional gene reporters, Truong et al. generated a novel cassette containing the coding sequence for a protein of interest along with requisite optimized sequences for nuclear RNA export and mRNA translation surrounded by optimized splice donor and splice acceptor sites (Figure 1 A-B)⁶. This cassette is then inserted into an exon of the parent gene allowing for transcription of a cistronic transcript encoding the parent gene and the reporter gene. This transcript is ultimately spliced to generate an untampered mature RNA sequence of the parent gene along with a separate transcript encoding the reporter gene. The authors utilized viral nuclear export sequences from the Mason-Pfizer monkey virus (MPMV) to trigger nuclear RNA export of the synthetic gene reporter RNA instead of inducing lariat formation and degradation as would normally occur with excised naturally encoded introns. The synthetic RNAs can then be translated into an effector protein of choice in a 5’-cap-independent manner or even exported out of the cell as an RNA reporter via engineered cell export. The authors have dubbed this new technology with the acronym INSPECT for Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts.

Figure 1. Methods to measure and regulate gene expression using splicing of synthetic RNA sequences.

(A) The Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts (INSPECT) technology by Truong et al.⁶ embeds a reporter construct within an artificial intron in a parent gene. This allows for dynamic monitoring of endogenous gene expression without perturbing nucleic acid sequence and an ability to link production of a gene of interest (GOI) from any host gene. (B) Details of the INSPECT technology and demonstration of its use to dynamically monitor activation of the T-cell receptor (TCR). The construct which includes the coding sequence of a GOI within an intron flanked by RNA nuclear export signals (NES) and splice donor and acceptor sites as well as an upstream IRES. The parent gene is transcribed along with the cistronic construct, each of which are then spliced to produced two distinct protein-encoding transcripts. The authors demonstrate the utility of this technique to monitor IL-2 protein production via production of luciferase or the transmembrane ion channel SCL5A5. In this manner, TCR activation can be monitored even by imaging with the use of radiolabeled iodine. (C) X^on technique by Monteys et al.¹³ whereby drug-regulated splicing is used for temporal regulation of gene expression. In this technology a minigene based on the gene SMN2, is placed upstream of a GOI. In the presence of orally administered medications which regulate SMN2 splicing, the GOI is in-frame and expressed. In the absence of drug treatment, a stop codon is present which prevents expression of the GOI. (D) Synthetic intron technique by North et al. whereby a synthetic intron is differentially unexcised depending on the presence or absence of oncogenic mutations in the core RNA splicing SF3B1 ¹⁴. Expression of therapeutic proteins in this manner may selectively deplete cancer cells based on the genotype of the spliceosome.

The authors first utilized this system to drive expression of luminescent proteins (such as NanoLuc luciferase) from the endogenous locus of the gene encoding IL-2. The INSPECT cassette encoding NanoLuc Luciferase was inserted into exon 3 of IL-2 in a T-cell cell line. The authors then demonstrated that activation of the T-cell receptor (TCR) resulted in dynamic, secreted, functional IL-2 protein (indicating that manipulation of the parent IL-2 gene did not perturb IL-2 expression or function) with concomitant production of NanoLuc luciferase signal (indicative of efficient nuclear export and translation of the synthetic RNA reporter from the IL-2 locus). In this manner, the authors demonstrated the ability of this approach to provide a real-time readout of endogenous gene expression that could be utilized to track gene expression by imaging-based methods. Taking this latter point further, the authors harnessed their technique to link expression of IL-2 to expression of the transmembrane sodium iodide symporter SLC5A5. SLC5A5 is normally only expressed on thyroid cells and can import radiolabeled iodide ¹³¹I⁻ into cells, which are then visualizable in vivo by SPECT based imaging.^{7, 8} In this manner, the authors were able to create an innovative method whereby T-cells can be imaged upon activation of the TCR. The ability to link activation of TCRs to production of an imageable protein provides the exciting possibility of inserting INSPECT cassettes into T-cell based cellular therapies, such as CAR-T cells, for real-time in vivo imaging of engineered T-cells in patients upon TCR activation.

As noted above, one important advantage of this new method is the ability to also monitor expression of non-protein coding RNAs. The authors demonstrated this approach via insertion of luciferase coding sequences into exons of the lncRNAs GUARDIN⁹ and NEAT1.¹⁰ They showed the dynamic activity of the reporter sequences as CRISPRi silencing of the lncRNA also silenced reporter protein expression. Importantly, NEAT1 is expressed as multiple distinct isoforms which differ in their subcellular localization.^{10, 11} By embedding INSPECT reporters into distinct exons of NEAT1, the investigators were able to read out expression of distinct mRNA isoforms of NEAT1, adding an additional layer of utility to their gene monitoring approach.

While the authors have focused their initial applications of the INSPECT approach to monitoring gene expression for basic science research and imaging, this technology could have therapeutic potential as well. Ultimately INSPECT links expression of an endogenous gene product to simultaneous generation of an additional gene of interest. As such, one could envision using this approach to eradicate cells with expression of oncogenic genes via generation of suicide genes or otherwise therapeutically targetable proteins.

Despite the strengths of the INSPECT approach, it is important to consider that the exact site of introduction of the reporter sequence is likely critical to success of this technology. First, the reporter is inserted via CRISPR/Cas9-mediated knockin which places some constraints on the precise location of reporter introduction. Given that the INSPECT approach inserts an artificial intronic sequence within an exon, it is possible that the site of sequence introduction could influence RNA splicing and/or expression of the parent gene. While these potential deleterious changes were not seen with the specific examples in the publication, it is possible that an artificial sequence could alter splicing regulatory sequences within an exon¹² and/or regulatory sequences if the reporter cassette is introduced near an intron/exon border.

The INSPECT approach is one of several recently reported technologies which harness the cell’s RNA splicing machinery to regulate gene expression using synthetic RNA sequences (Figure 1 C-D). For example, Monteys et al. recently described an approach using drug-regulated splicing events to temporally control expression of gene products using chemical matter (Figure 1 C).¹³ Specifically, they harnessed portions of SMN2 where several orally administered drugs have been developed to regulate SMN2 productive splicing for the treatment of spinal muscular atrophy.¹³ An SMN2 minigene was utilized to control the frame of the coding sequence of a gene of interest such that in the presence of drug, SMN2 splicing generates an inframe protein coding mRNA. In the absence of drug, the SMN2 minigene contains a stop codon and prevents translation of the gene of interest. This technique was used for timed regulation of the expression of fluorescent proteins, Cas9, and erythropoietin in animal models in vivo.¹³

More recently, North et al. developed a technique whereby synthetic intronic sequences which uniquely respond to the presence of oncogenic mutations in the core RNA splicing factor SF3B1 are used to regulate gene expression in a genotype dependent manner (Figure 1 D).¹⁴ This synthetic intron technology thereby can be utilized to express therapeutic proteins to eliminate cancer cells containing specific genetic alterations in the splicing machinery.

Overall, each of these technologies noted above demonstrates the utility and versatility of RNA to regulate and monitor gene expression in a gene, genotype, and/or temporal manner^13-15. Besides the clear differences in goals of the technologies reported by Truong et al., Monteys et al., and North et al., it is important to note that the INSPECT approach necessitates insertion into the genome, while the other two technologies can function via expression vectors that do not integrate into the genome. While these techniques will have a myriad of applications in cell biological and preclinical studies, demonstrating utility and delivery in vivo will be important next steps. In parallel, it will be particularly exciting to consider potential use of these approaches in cell therapeutics where they can be introduced ex vivo and for potential gene expression regulation and cell therapy monitoring in vivo.