What Is Non-Coding RNA?
Non-coding RNA (ncRNA) is transcribed from the regions of the genome that are not responsible for protein coding. This function falls to messenger RNA (mRNA).
Historically, non-coding regions of the genome were thought to harbor “junk DNA,” and it wasn’t even thought that they were transcribed. As sequencing technologies advanced, scientists realized that ncRNA was transcribed from some of these “junk DNA” regions, but it was not known to have any biological purpose.
However, as research continues, we have learned more about ncRNA’s role in functions such as regulating gene expression, directing cell proliferation and cell death, and the critical role these ncRNAs play in developmental timing.
We don’t have a complete list of ncRNA types or roles, but scientists are learning more each day. In this article, we will explore some of the types of ncRNA that technologies at Psomagen can help to explore.
Short Non-Coding RNA
Non-coding RNA is split into two subgroups. The first, short non-coding RNA (sncRNA), consists of non-coding RNA molecules of fewer than 200 nucleotides in length, including:
Transfer RNA (tRNA), a key component in translation and protein synthesis.
tRNA provides the link in piecing together amino acid chains from the instructions found in the genetic code. When a tRNA pairs with the correct RNA codon, the correct amino acid is added to the chain.
tRNA malfunctions impact the formation of tRNA and, in turn, the proteins they compile. This is a major area of neurological research, where tRNA dysregulation impacts many brain disorders.
In 2015, for example, researchers discovered a genetic mutation in three siblings with Leigh syndrome. This mutation led to a reduction in steady state levels of tRNATrp, which in turn led to decreased protein synthesis and decreased levels of proteins responsible for mitochondrial respiration. This study linked this genetic mutation with Leigh syndrome partnered with severe multi-organ disease.
Transfer RNA-Derived Fragments (tRFs) are even smaller fragments derived from mature tRNAs or pre-tRNAs, usually 14-32 nt in length. Like tRNA, they are involved in protein translation, gene expression, and cell cycles.
microRNA (miRNA), with roles in regulatory pathways and gene silencing.
These functions are commonly seen in organism development, such as cardiac development and cardiomyocyte proliferation processes. miRNA can silence genes at the appropriate time in development. When this process is disrupted, by keeping a gene on longer than necessary or by turning off a necessary gene, development irregularities can occur. microRNA-9, for example, is a key component of neural cell development and regulation, and is abundant in neural progenitor cells. Downregulation of miR-9 is implicated in a subset of schizophrenia cases.
Dysregulation of miRNA has a proven impact on disease development. In breast cancer, for example, miRNA methylation can lead to chemotherapy and radiation resistance. Understanding these changes is an important step toward personalized treatments in disease states.
miRNAs are one of the most prolific types of ncRNA. Thousands of miRNAs have been reported in recent years, with research uncovering more all the time — so much so that the 2024 Nobel Prize in Physiology or Medicine was awarded to Doctors Victor Ambrose and Gary Ruvkin for their discovery of miRNA, a breakthrough that occurred over two decades ago.
Small nucleolar RNA (snoRNA), with a role in overseeing chemical changes occurring in other types of RNA.
This includes ribosomal RNA (rRNA), tRNA, and small nuclear RNA (snRNA). Before pre-rRNA becomes a mature rRNA molecule, snoRNA guides a series of modifications, including methylation and pseudouridylation. Faulty snoRNA functioning can result in impaired pre-rRNA maturation or a reduction in the number of functional ribosomes, which in turn reduces protein synthesis. In Prader-Willi syndrome, for example, a snoRNA gene has been identified as critical to the disease, and the most likely cause of neonatal Prader-Willi syndrome lethality in mice.
snoRNA has potential as a noninvasive biomarker in several types of cancer. Their role in tumorigenesis makes them a strong indicator of patient prognosis. In clear cell renal cell carcinoma (ccRCC), researchers created a six-snoRNA signature and stratified patients based on risk level. Further testing revealed that this signature was a better indicator than existing clinical analyses in ccRCC. Other studies have found links between snoRNA expression and prognosis in ovarian cancer, melanoma, and other cancers.
Small interfering RNA (siRNA), with roles in cell defense and gene regulation.
siRNA degrades mRNA after transcription, preventing it from being translated into proteins. This mechanism, called RNA interference (RNAi), can be induced via synthetic siRNA for therapeutic purposes. In gastric cancer research, siRNA has a proven role in diminishing cancer cell proliferation and inducing chemosensitivity to prevent tumor progression. The challenge, in gastric cancer and other oncology research projects, lies in designing effective delivery that keeps siRNA stable until it reaches the cancer cells.
siRNA functions naturally in eukaryotic organisms, particularly in immune response. In plants, siRNA has been identified as a major factor in controlling intracellular nucleotide-binding leucine-rich repeat protein (NLRs) levels. NLRs are activated when plants detect pathogen-derived effectors, and are an important role in immune response. When dysregulated, elevated NLR levels can lead to developmental arrest, loss of fitness, and runaway cell death.
Interestingly, siRNA was originally discovered in petunia by a scientist who was trying to deepen the purple color of the blossoms — instead, they lost all pigment.
Small nuclear RNA (snRNA), important to the splicing process of pre-mRNA.
In this process, introns are removed. The remaining exons can then be coded into proteins. Splicing errors can negatively impact cell health and function. Abnormal splicing in the SLC26A4 gene, for example, can cause hereditary hearing loss. This study identified 11 U1 snRNA mutations that caused abnormal SLC26A4 splicing. By modifying the mutated U1 snRNA, researchers were able to fix abnormal splicing, suggesting that this snRNA is a strong therapeutic candidate.
Piwi-interacting RNA (piRNA), found in animal cells, is a part of regulatory and immune response.
piRNAs get their name from the PIWI proteins they bind with, which are implicated in stem cell and germ cell formation, including processes like spermatogenesis in mice and sex-specific gene regulation in fruit flies.
Recent research has investigated the role of piRNAs with aberrant expression in cancer stem cells (CSCs). piRNA-823, which is overexpressed in luminal breast cancer cells, was shown to alter DNA methylation profiles and promote cancer cell growth and colony formation. It was also found to promote tumor regeneration.
This regulatory role makes piRNA a promising therapeutic target in many oncology research areas. In the study above, piRNA-823-targeting gene therapy successfully inhibited luminal breast cancer tumorigenesis and tumor growth in a mouse model.
Small activating RNA (saRNA) regulates gene expression by binding to promoter regions.
saRNA is rather like the opposite of miRNA. Scientists have successfully used saRNA to activate genes in cultured cells, making these molecules a potential therapeutic mechanism and biotechnology target.
Small rDNA-derived RNA (srRNA).
srRNA have been shown to play a role in DNA damage repair in Neurospora crassa. Researchers are currently exploring potential applications of srRNA in vaccine development and gene therapy.
Long Non-Coding RNA
Long non-coding RNA (lncRNA) are non-coding RNA molecules of over 200 nucleotides in length. These molecules are involved in normal organism development, and like some sncRNAs, they play a role in regulating gene expression. lncRNAs are often categorized based on the location of the genomic region responsible for transcribing them.
Intergenic lncRNAs (lincRNAs), transcribed from genomic regions between genes.
Not all lincRNAs have been characterized. However, the ones that have been researched give us a glimpse into lincRNA’s diverse range of functions. These functions include epigenetic regulation, impacting transcriptional activation through modifying chromatin states, and acting as decoys for proteins and other RNAs.
In some species, including Brassicaceae plants and yeast, scientists have completed deeper research into profiling the role of lincRNA. Some molecules we currently categorize as lincRNA have been found to encode peptides, suggesting a need to recategorize some lincRNAs as coding molecules.
Intronic lncRNAs, transcribed from introns within protein-coding genes.
These molecules may influence transcription, splicing, and RNA processing events. Like lincRNAs, intronic lncRNA is a large group with varying functions. They have a clear correlation with the development of many cancers. Intronic lncRNA has been investigated for its suspected role in metastasis of pancreatic cancer, particularly in aggressive pancreatic ductal adenocarcinoma. Differential expression of three intronic lncRNAs was found in metastatic samples.
Enhancer RNAs (eRNAs), transcribed from enhancer regions of the genome.
eRNA transcription has a strong correlation with gene enhancer activity. They can work with gene enhancers to impact chromatin accessibility, histone modifications, and gene expression. Although this function is not yet well understood, researchers suspect that eRNA plays a role in the formation of the “chromatin loop,” a mechanism by which enhancers are brought to their target gene.
eRNA is found at a higher concentration in many types of cancer. In glioma research, eRNAs are associated with immune dysfunction in the tumor microenvironment. Scientists believe that targeting eRNA-regulated immune-related genes could be an effective therapeutic option.
Promoter-associated RNAs (paRNAs), transcribed from genetic regions near gene promoters.
These RNAs can influence transcription and chromatin structure.
Telomere repeat-containing RNAs (TERRA), transcribed from telomeric regions of chromosomes.
Telomeres are the repetitive ends of a chromosome that protect chromosome structure. Some research has indicated that TERRA may help regulate the length of telomere repeats. In ICF, a rare recessive disease occurring in humans, research has found elevated TERRA levels and abnormally shortened telomeres.
Ribosomal RNAs (rRNAs), responsible for protein synthesis in ribosomes.
rRNA binds tRNA and mRNA, helping to translate the mRNA sequencing into an amino acid chain. rRNA makes up about 80% of an RNA sample. Most RNA sequencing projects involve an rRNA depletion step, increasing the amount of relevant sequencing reads when analyzing mRNA, sncRNA, or lncRNA.
Circular RNAs (cirRNAs), single-stranded loops of RNA formed when the 3’ and 5’ ends of the RNA are joined.
cirRNAs were first discovered as pathogens in plants. However, they are found in many organisms. Their aberrant expression has been linked to many disease states, including cancers, autoimmune disorders, and cardiovascular disease.
cirRNAs act as “miRNA sponges,” binding with miRNA molecules and preventing them from interacting with mRNAs. In non-small cell lung cancer, ciRS-7 has binding sites for miR-7. Patients with highly expressed ciRS-7 and lowly expressed miR-7 exhibited larger tumor size and severe metastasis in the lymph nodes, as well as advanced histopathological grading of their cancer.
cirRNAs have also been studied for their interactions with proteins. These interactions have been implicated in apoptosis, tumor growth and suppression, metastasis, inflammation, and other critical biological functions. These interactions are not yet well understood or comprehensively studied, and we can expect more insights as research progresses.
Technologies for Non-Coding RNA Research
Several options now exist for exploring non-coding RNA, whether on its own or as part of the larger transcriptomics landscape.
Total RNA sequencing
Total RNA sequencing measures genes and transcripts present in all coding and non-coding molecules in a transcriptome. This offers a high-level view of all RNA content, including the types listed above. Strand-specific total RNA sequencing makes it possible to identify overlapping transcripts and genes.
Small RNA sequencing
Small RNA sequencing is a more focused option than total RNA sequencing, analyzing only the small non-coding RNA fragments in a sample. Small RNA seq enriches a total RNA sample for sncRNA molecules, including the types discussed in this article. Options for tRNA and YRNA blocking generate more microRNA reads, if that’s the researcher’s area of interest.
Low-input RNA sequencing
Many service companies now offer low-input RNA sequencing options. This overcomes a significant hurdle that RNA researchers have faced for years: the large sample size necessary for RNA sequencing. With low-input RNA sequencing, less than 10ng of sample can still deliver meaningful results about coding and non-coding regions. This option is a common choice for assessing rare RNA subtypes of cell populations, where obtaining a large sample may be very difficult.
Single-cell transcriptomics
Single-cell sequencing goes beyond traditional RNA sequencing methods by providing a transcriptomic profile of each individual cell, rather than a bulk readout. Not all single-cell approaches will focus on non-coding RNA. However, specialized kits and workflows do make this possible on some platforms.
Short and long non-coding RNAs play an essential role in regulating biological processes. These molecules, once thought to be transcriptional noise, have emerged as key players in gene expression, cellular signaling, and disease pathology. Advances in sequencing technologies have increased our understanding of these RNAs, revealing their diverse functions and mechanisms.
By leveraging emerging technologies like single-cell transcriptomics and AI-driven analysis, scientists are poised to explore their roles with unprecedented resolution. Understanding non-coding RNAs enriches our knowledge of fundamental biology and creates new possibilities for therapeutic development in precision medicine.
