RNA-mediated oligonucleotide Annealing, Selection, and Ligation with Next-Gen sequencing (RASL-seq) is a targeted high-throughput RNA-seq-based technology that has contributed to diverse biological discoveries from the identification of novel cancer therapeutics to the broad importance of alternative splicing in human health and disease.
The third article in our RASL-seq mini-series looks at how researchers have used this technology to advance our knowledge of fundamental biological and disease processes to accelerate therapeutic discoveries and improve human health ultimately.
RASL-seq proof-of-concept in cancer therapeutics
With any novel technology, researchers must extensively validate the method with carefully designed experiments addressing complex questions to build trust before its widespread uptake by the research community.
RASL-seq was no exception, and to prove its worth in large-scale drug screens, the inventors of this technology aimed to find new therapeutics capable of blocking the expression of a cancer-causing gatekeeper called the androgen receptor (AR) that drives tumor growth in the prostate. The AR is a compelling therapeutic target because when it is overexpressed, it drives the expression of other key genes involved in the progression of prostate cancer and affects the response of tumors to standard treatments (Westaby et al., 2022).
To find these potential therapeutics, they treated prostate cancer cells with over 4,000 compounds and used RASL-seq to detect the transcriptional effects of each treatment. They evaluated the expression of around 70 androgen receptor-responsive genes and included 30 genes not typically influenced by the androgen receptor as controls (Li et al., 2012).
This targeted strategy identified one compound that repressed AR expression, limited the expression of AR-responsive genes, and ultimately stopped prostate cancer cells from proliferating. Notably, the compound was effective even in prostate cancer cells resistant to other standard prostate cancer therapies (Li et al., 2012).
Alongside its use in large-scale compound screens to generate targeted gene expression profiles, the paired probe design of RASL-seq also lends itself to the sensitive detection of alternative splicing events in response to therapeutics, disease progression, or genetic mutations.
RASL-seq detects RNA splicing differences in metastatic breast cancer
RNA splicing is the process of excluding introns from pre-mRNA to produce mature mRNA containing only exons. Frequently, different exons can be included or skipped in an mRNA transcript to generate alternatively spliced mRNA isoforms with potentially different mRNA stabilities or protein products.
mRNA splicing is crucial to disease as it influences all aspects of the progression of cancer, from its severity to the sensitivity of a tumor to treatment (Bonnal, López-Oreja and Valcárcel, 2020).
Therefore, researchers used over 5,000 RASL-seq probe pairs spanning known splice junctions to determine if highly metastatic breast cancer cells had different mRNA splicing than breast cancer cells with lower metastasis (Oh et al., 2021).
The researchers found that differences in alternative splicing were widespread between the two types of cancer cells. They also discovered that excluding a single exon in a crucial cancer-causing gene resulted in more stable mRNA, which was linked to severe metastasis and reduced patient survival.
RASL-seq in other therapeutic and diagnostic areas
Furthermore, the power of RASL-seq for detecting alternative splicing is not limited to the oncology field. Researchers have applied it to other disease areas such as neurodegeneration to determine critical genes involved in splicing machinery, which can cause the fatal motor neuron disease amyotrophic lateral sclerosis when mutated (Sun et al., 2015).
RASL-seq has even been adapted to provide a highly-sensitive and scalable diagnostic tool for clinicians to detect SARS-CoV-2 directly on nasopharyngeal swabs without relying on time-consuming and expensive nucleic acid purification or reverse transcription necessary in other methods (Credle et al., 2021).
New opportunities for ultra-high-throughput gene expression profiling
While RASL-seq remains the method of choice for the high-throughput quantitative detection of known alternative splicing events, other technologies have superseded this method for high-throughput gene expression profiling in drug discovery pipelines and address some critical limitations of RASL-seq (Alpern et al., 2019).
For example, instead of being limited to the detection of a maximum of 500 genes as with RASL-seq, new 3’ bulk mRNA-seq technologies such as MERCURIUS™ bulk RNA barcoding and sequencing (BRB-seq) and MERCURIUS™ DRUG-seq combine ultra-scalability to thousands of samples and the generation of expression profiles for around 20,000 genes in the human genome.
Please contact us or see the other articles in our mini-series to learn more about RASL-seq, MERCURIUS™ BRB-seq, and MERCURIUS™ DRUG-seq.
References
- Alpern, D. et al. (2019) ‘BRB-seq: Ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing’, Genome Biology, 20(1), pp.1-15. Available at: https://doi.org/10.1186/s13059-019-1671-x.
- Bonnal, S.C., López-Oreja, I. and Valcárcel, J. (2020) ‘Roles and mechanisms of alternative splicing in cancer—implications for care’, Nature Reviews Clinical Oncology, 17(8), pp.457-474. Available at: https://doi.org/10.1038/s41571-020-0350-x.
- Credle, J.J. et al. (2021) ‘Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping’, Modern Pathology, 34(6), pp.1093-1103. Available at: https://doi.org/10.1038/s41379-020-00730-5.
- Li, H. et al. (2012) ‘Versatile pathway-centric approach based on high-throughput sequencing to anticancer drug discovery’, Proceedings of the National Academy of Sciences, 109(12), pp. 4609–4614. Available at: https://doi.org/10.1073/pnas.1200305109.
- Oh, J. et al. (2021) ‘Widespread alternative splicing changes in metastatic breast cancer cells’, Cells, 10(4), p.858. Available at: https://doi.org/10.3390/cells10040858.
- Sun, S. et al. (2015) ‘ALS-causative mutations in FUS/TLS confer gain and loss of function by altered association with SMN and U1-snRNP’, Nature Communications, 6(1), p.6171. Available at: https://doi.org/10.1038/ncomms7171.
- Westaby, D. et al. (2022) ‘A new old target: androgen receptor signaling and advanced prostate cancer’, Annual Review of Pharmacology and Toxicology, 62, pp.131-153. Available at: https://doi.org/10.1146/annurev-pharmtox-052220-015912.
