Structure of lncRNAs
Once dismissed as “junk”, long noncoding RNAs (lncRNAs) are now recognized as versatile regulators in cellular biology. They are defined as RNA transcripts longer than 200 nucleotides that do not encode proteins. They participate in a wide array of biological processes through different mechanisms.
lncRNAs exhibit a size that spans from 200 bp to over 100 kb in length. They are often transcribed by RNA polymerase II, 5′-capped, and polyadenylated, although a significant subset of lncRNA lack poly(A) tails. Their secondary and tertiary structures are complex, with domains that fold independently and contain conserved motifs such as helices, junctions, internal loops, and terminal loops, which serve as independent functional units for their interactions with DNA, RNA, and proteins.
Compared to protein-coding RNAs, lncRNAs contain a higher proportion of non-canonical splice sites (e.g., GC–AG), resulting in less efficient but increased alternative splicing. There are tens of thousands of lncRNA genes in the human genome – current annotations list over 20,000 lncRNA genes, with some databases estimating >90,000 lncRNA. It is estimated that ~75% of the human genome is transcribed into non-coding RNAs, and lncRNAs are a major component of this “dark” transcriptome.
Biological function of lncRNAs
lncRNAs participate in gene regulation at almost every level. They can act in the cytosol to stabilize mRNAs, modulate translation, or sponge microRNAs, affecting gene expression post-transcriptionally. They can enhance or repress transcription. A well-known example is MALAT1, a lncRNA that in the cytoplasm can bind to ribosomal proteins and translation factors, modulating the translation process.
In the nucleus, lncRNAs associate with chromatin and the transcription machinery – they recruit histone modifiers, act as enhancers, or influence alternative splicing of pre-mRNAs. They often serve as scaffolds that bring together multiple proteins into ribonucleoprotein complexes. For instance, lncRNA NEAT1 forms the scaffold for nuclear paraspeckle bodies by binding multiple RNA-binding proteins.
Though less common, lncRNAs can form RNA-DNA hybrids (R-loops) or triple-helices, allowing them to directly pair with complementary DNA sequences. This can guide lncRNAs to specific genomic sites or modulate local DNA replication and repair. While evidence of direct RNA–DNA triplex function is emerging, it suggests another layer by which lncRNAs influence gene expression and chromatin architecture.
lncRNAs as Biomarkers
One of the most promising aspects of lncRNAs is their potential use as biomarkers for disease diagnosis, prognosis, and even as therapeutic monitors. Their tissue specificity and dysregulation in disease make lncRNAs attractive indicators for a variety of pathologies, including cancer. For example, a lncRNA known as HOTAIR is overexpressed in breast cancers and promotes metastasis by reprogramming chromatin states. It has been established that high levels of UCA1 in urine can indicate presence of bladder tumors.
Dysregulated lncRNA have also been found in Alzheimer’s disease, where lncRNA BACE1-AS regulates the BACE1 mRNA involved in amyloid-beta production. lncRNA also play an important role in regulating the expression of inflammatory mediators and are being studied as potential therapeutic targets for controlling chronic inflammation.
lncRNAs often circulate in body fluids (blood, urine, saliva) within exosomes or ribonucleoprotein complexes, which can protect them from degradation. Notably, some lncRNAs are exceptionally stable in blood – for example, the prostate cancer lncRNA PCA3 is detectable in urine and is resistant to nuclease digestion and freeze-thaw cycle. This opens the door for development of minimally invasive tests (similar to how cell-free DNA or miRNAs are used in liquid biopsies).
Beyond being static biomarkers, lncRNAs are being investigated also in therapeutic contexts, either as drug targets themselves or as indicators of therapeutic response.
Next-Generation Sequencing (NGS) for lncRNA Research
Studying lncRNAs requires transcriptome-wide profiling methods due to their often-low abundance and complex splicing patterns. NGS technologies have become indispensable for identifying and quantifying lncRNAs.
From a technical standpoint, stranded total RNA sequencing, such as the one enabled by the NEXTFLEX Rapid Directional RNA-Seq Kit 2.0 is the default recommended workflow. Strand-specific library construction is critical for distinguishing overlapping sense and antisense transcripts, a common feature of lncRNAs. Using this kit with rRNA depletion (for example with the NEXTFLEX RiboNaut rRNA depletion kit upstream or with CRISPR-based depletion downstream) will allow capturing both polyadenylated and non-polyadenylated lncRNA species in an unbiased way. This workflow allows incorporating low or high input RNA from high quality and degraded samples, and together with the NEXTFLEX UDI-UMI barcodes researchers can sequence deeper without being affected by PCR duplicates.
Next-generation sequencing of lncRNAs plays a crucial role in functional genomics by providing comprehensive insights into lncRNA expression patterns and interactions. It enables the identification of lncRNA targets and their regulatory networks, further elucidating their functions and implications in various biological processes. It is used in conjunction with high-throughput screening and CRISPR-based genome editing, to investigate the roles of lncRNAs.
Conclusion
Long noncoding RNAs are key players in gene regulation and hold great promise as biomarkers for disease diagnosis and prognosis. Their structural complexity and diverse functions necessitate advanced sequencing methods to unravel their biology. Revvity provides researchers with the tools to explore the lncRNA landscape in detail, accelerating discoveries that could translate into critical innovations.
References
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- Zhang, Y., et al. Roles of long noncoding RNAs in human inflammatory diseases. Cell Death Discov. 10, 235 (2024).
- Marchese, F.P., et al. Long non-coding RNAs: definitions, functions, challenges and opportunities. Nat Rev Mol Cell Biol 24, 245–262 (2023).
- Badowski, C., He, et al. Blood-derived lncRNAs as biomarkers for cancer diagnosis: the Good, the Bad and the Beauty. npj Precis. Onc. 6, 40 (2022)
- Statello, L., et al. Gene regulation by long non-coding RNAs and its biological functions. Nat Rev Mol Cell Biol 21, 96–114 (2020). Quinn, J.J., Chang, H.Y. Unique features of long non-coding RNA biogenesis and function. Nat Rev Genet 17, 47–62 (2021). Kopp, F., Mendell, J.T. Functional classification and experimental dissection of long noncoding RNAs. Cell 172, 393–407 (2020).
