Non-Coding RNA Research and Technologies

Non-coding RNAs (ncRNAs) do not code for proteins but play important roles in numerous cellular processes including gene silencing, developmental pathways, and more. There are numerous types including microRNA (miRNA), long ncRNA (lncRNA), circular RNA (circRNA), and more. In this article, we discuss innovative ncRNA research and explore recent technological advancements that improve the study of ncRNAs.

Nobel Prize for MicroRNA Discovery
This week, Victor Ambros and Gary Ruvkun were awarded the 2024 Nobel Prize in Physiology or Medicine for their discovery of microRNA and its role in controlling gene activity. MicroRNAs are small RNA molecules that regulate gene expression after transcription¹. Ambros and Ruvkun’s research on C. elegans led to the identification of lin-4, a gene producing a short RNA molecule that regulates another gene, lin-14, by blocking its protein production^2,3. This discovery introduced a new understanding of gene regulation, showing that microRNAs bind to complementary sequences in mRNA and prevent protein synthesis. Their work was initially met with skepticism, but several years later, another microRNA, let-7, was identified and found to be highly conserved across species. This helped establish the acceptance of microRNA regulation in multicellular organisms. Ambros and Ruvkun's breakthroughs helped scientists understand new ways genes are regulated after transcription, revealing a key process that is integral to complex life forms.

X Chromosome Inactivation and Autoimmunity
Another expert studying the impact of important ncRNAs is Montserrat C. Anguera Ph.D., Associate Professor in the Department of Biomedical Sciences at the University of Pennsylvania School of Veterinary Medicine. Anguera's lab is largely focused on studying X chromosome inactivation (XCI) in the immune system. "We want to understand how the X chromosome —both the genetics and the epigenetics of the X chromosome—contributes to female bias diseases that involve the immune system,” stated Anguera. Historically, sex hormones were thought to explain this difference, but Anguera's group is studying how gene expression may play an important role.

During XCI, one of the two X chromosomes in XX individuals is silenced to balance gene expression. This process is heavily dependent on Xist, a long non-coding RNA (lncRNA). “Xist is really a fascinating RNA molecule because it works in cis, and it can transcriptionally silence most of the chromosome from where it's expressed,” explained Anguera. In particular, it silences most of the X chromosome by recruiting proteins and adding epigenetic modifications that ensure the silenced X remains inactive. However, Anguera's lab discovered that some immune cells, like T cells and B cells, do not always maintain the typical markers of XCI seen in other cell types⁴. In these immune cells, Xist is expressed but it does not localize to the inactive X chromosome, and heterochromatic marks that typically overlap with Xist RNA are absent. "It's almost like the inactive X is not as tightly compact and it's amenable for parts of it to get reactivated in these immune cells, if necessary," noted Anguera. These findings suggest that partial reactivation of the inactive X could contribute to females' heightened immune response and increased susceptibility to autoimmune disorders like lupus.

Since her landmark study into this process, Anguera has continued investigating the role of Xist deletion in B cells and its connection to the development of systemic lupus erythematosus (SLE)⁵. After developing a mouse model with B cell-specific deletion of the Xist gene, Anguera’s group observed that some female mice with this deletion spontaneously developed SLE-like symptoms, such as the production of disease-specific autoantibodies, glomerulonephritis, and expansion of activated B cell subsets. Furthermore, the severity of SLE-like disease increased in these mice when exposed to pristane, a chemical that induces lupus-like symptoms. The study demonstrated that impaired XCI in B cells can contribute to the female bias observed in SLE by upregulating proinflammatory X-linked genes, such as Tasl, a key component of the TLR7 signaling pathway linked to SLE pathogenesis.

In another recent study, the lab explored the role of NF-κB signaling in maintaining X chromosome inactivation in T cells following activation⁶. They revealed that the inactive X chromosome in unstimulated T cells is mostly dosage-compensated with specific epigenetic modifications, particularly H3K27me3, but lacks others like H2AK119-ubiquitin. After T cell activation, these marks accumulate, and NF-κB signaling, triggered through T cell receptor (TCR) engagement, is important for this process. The disruption of NF-κB signaling in both mouse models and human cells impairs the localization of Xist RNA and alters the expression of certain X-linked genes. This disruption may have significant implications for understanding sex differences in immune responses and the development of autoimmune diseases.

Technologies and Advances
In her ncRNA research, Anguera explained that her group uses a variety of techniques. The most beneficial has been RNA FISH (fluorescence in situ hybridization). Despite being an older method, RNA FISH was essential for identifying that Xist RNA is expressed but not properly localized on the inactive X chromosome in immune cells. Anguera noted that this discovery would likely be missed by more modern methods like RNA-seq and microarrays.

One major technological advancement in ncRNA research has been the ability to directly sequence RNA. “Prior to nanopore sequencing, there was no way for scientists to directly analyze RNA molecules with sequencing technologies,” explained Libby Snell, Ph.D., Director of RNA and cDNA Sample Technology at Oxford Nanopore Technologies. “Instead, RNA had to be converted to cDNA, and the information gleaned from cDNA analysis was used to infer knowledge about the original RNA. That step adds cost and complexity, but more importantly, it can introduce errors and strip out useful information. Furthermore, the act of converting RNA to cDNA can suffer from certain biases due to the nature of the sequence within the RNA transcripts.”

Snell highlighted recent improvements in their RNA analysis tools, specifically their newest RNA004 chemistry, which offers 98.8% single-read accuracy and can generate up to 20-30 million reads per sample. These developments have enhanced the detection of ncRNAs, particularly long non-coding RNAs (lncRNAs) that were previously undetectable. Snell also emphasized that the ability to study RNA directly, without converting it to cDNA, has opened up new possibilities for scientific research. This direct approach has led to the discovery of numerous novel lncRNAs, expanding the catalog of known ncRNAs. These discoveries are helping scientists uncover the functions of these molecules, with recent advances seen in fields like precision oncology, parasite biology, and extracellular vesicle analysis. However, Snell acknowledged that one of the biggest challenges in ncRNA research is making sense of novel findings that deviate from what was previously inferred using cDNA-based analysis. Direct RNA sequencing is helping to address this by providing more comprehensive and accurate RNA data.

“Next-generation sequencing has been a huge step forward in understanding the role of RNA in biology, and I believe with long-read, direct RNA sequencing, we are now taking the next big leap by empowering scientists with novel analysis possibilities, including nucleoside modifications within the context of full-length transcript information,” stated Snell. “The discoveries made today will become the foundation for understanding and interpreting non-coding RNAs in the decades ahead.”

References

1. Shang R, Lee S, Senavirathne G, Lai EC. microRNAs in action: biogenesis, function and regulation. Nature Reviews Genetics. 2023;24(12):816-833. doi:https://doi.org/10.1038/s41576-023-00611-y
2. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin4 encodes small RNAs with antisense complementarity to lin14. Cell. 1993;75(5):843-854. doi:https://doi.org/10.1016/0092-8674(93)90529-Y
3. Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin14 by lin4 mediates temporal pattern formation in C. elegans. Cell. 1993;75(5):855-862. doi:https://doi.org/10.1016/0092-8674(93)90530-4
4. Wang J, Syrett CM, Kramer MC, Basu A, Atchison ML, Anguera, Montserrat C. Unusual maintenance of X chromosome inactivation predisposes female lymphocytes for increased expression from the inactive X. Proceedings of the National Academy of Sciences. 2016;113(14):E2029-E2038. doi:https://doi.org/10.1073/pnas.1520113113
5. Lovell CD, Jiwrajka N, Amerman HK, Cancro MP, Anguera, Montserrat C. Xist Deletion in B Cells Results in Systemic Lupus Erythematosus Phenotypes. bioRxiv. Published online January 1, 2024:2024.05.15.594175. doi:https://doi.org/10.1101/2024.05.15.594175
6. Forsyth KS, Toothacre NE, Jiwrajka N, et al. NF-κB Signaling is Required for X-Chromosome Inactivation Maintenance Following T cell Activation. bioRxiv. Published online January 1, 2024:2024.02.08.579505. doi:https://doi.org/10.1101/2024.02.08.579505