A majority of the human genome is made up of non-protein coding DNA. It infers that such sequences are not commonly employed to encode for a protein. However, even though these regions do not code for protein, they have other functions and carry necessary regulatory information.They can be classified based on the size of the ncRNA. Small noncoding RNA is usually categorized as being under 200 bp in length, whereas long noncoding RNA is greater than 200bp. In addition, they can be categorized by their function within the cell; Infrastructural and Regulatory ncRNAs. Infrastructural ncRNAs seem to have a housekeeping role in translation and splicing and include species such as rRNA, tRNA, snRNA.Regulatory ncRNAs are involved in the modification of other RNAs.
RNA Classification Long non-coding RNA Long non-coding RNA (LncRNA) are a type of RNA which is usually defined as transcripts which are greater than 200 base-pairs in length and not translated into proteins. This limitation distinguishes lncRNA from small non-coding RNAs which encompasses microRNAs (miRNAs), small interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), and other short RNAs. Long non-coding RNAs include lincRNAs, intronic ncRNAs, circular and linear ncRNA.
Long intergenic Non-coding RNA Long intergenic Non-coding RNA (LincRNA) is defined as RNA transcripts that are longer than 200 nucleotides. These RNAs must not have open reading frames that encode proteins. The term “intergenic” refers to the identification of these transcripts from regions of the genome that do not contain protein-encoding genes. LncRNAs also contain promoter - or enhancer-associated RNAs that are gene proximal and can be either in the sense or antisense orientation.
Circular RNA Circular RNA (CircRNA) are a novel class of endogenous noncoding RNAs and are characterized by their covalently closed loop structures. This class of ncRNA does not have a 5’ cap or 3’ Poly A tail. It has been hypothesized that cirRNAs may function as potential molecular markers for disease diagnosis and treatment and play an important role in the initiation and progression of human diseases.
Small non-coding RNA Small non-coding RNA (sncRNA) are a type of RNA. which is usually defined as transcripts which are lesser than 200 base-pairs in length and not translated into proteins. This limitation distinguishes sncRNA from lncRNA. This class includes but is not limited to microRNAs (miRNAs), small interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), and other short RNAs.
microRNA microRNA (miRNA) plays an important role in regulating gene expression. Majority of miRNAs are transcribed from DNA sequences into primary miRNAs. These primary miRNAs are further processed into precursor miRNAs, and finally into mature miRNAs. The miRNAs in most cases interact with the 3’ UTR region of target to induce mRNA degradation and translational repression. Interactions of miRNAs with other regions, including the 5’ UTR, coding sequence, and gene promoters have also been reported. Under certain conditions, miRNAs are also able to activate translation or regulate transcription, but this is dependent on factors such as location of the effect. This process of interaction is very dynamic and dependent on multiple factors.
Ribosomal RNA Ribosomal RNA (rRNA) includes non-coding RNAs that play essential roles in rRNA regulation. Ribosomal RNA (rRNA) takes part in protein synthesis. Occasional RNA molecules act catalytically, as RNA enzymes (ribozymes) or take part in protein export. The most important ribozyme is the major rRNA of the large subunit of the ribosome (28s rRNA in eukaryotes). It is now accepted that 28S rRNA catalyzes the critical step in polypeptide synthesis in addition to playing a major structural role.
Small nuclear RNA and small nucleolar RNA Small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA) are widely known to guide the nucleotide modifications and processing of rRNA. Both snRNA and snoRNA are categorized into a class of small RNA molecules that are present in the nucleus. However, they vary a lot by function. snRNA are 80-350nucletides long while snoRNA are 80-1000 nucleotides long in yeast. snRNA plays a critical role in regulating the pre-mRNA silencing. On the other hand, snoRNAs are involved in mRNA editing, modification of the rRNA and tRNA, and genome imprinting. Major function of snoRNA includes the maturation of rRNA during ribosomal formation. Small nuclear and small nucleolar RNAs are critical components of snRNPs and snoRNPs and play an essential role in the maturation of, respectively, mRNAs and rRNAs within the nucleus of eukaryotic cells. Both snRNA and snoRNA are involved in modifying RNA just after transcription. snRNA can be found in splicing speckles and Cajal bodies of the nucleus of the cell.snRNA and snoRNA requires a phosphorylated adaptor for nuclear export (PHAX) to get transported to the site of action within the nucleus.
Transfer RNA Transfer RNA (tRNA) helps decode a messenger RNA sequence into a protein. They function at specific sites within the ribosome during translation (the process going from code to protein). Within the mRNA molecule we have three nucleotides in length codons. These codons all have a unique universal code which represents a particular amino acid. tRNAs can be classified as an adaptor molecule, being typically 76 to 90 nucleotides in length.
History Non-coding RNA DNA purification in 1869 by Dr. Friedrich Miescher’s, from salmon sperm and pus cells guided the scientists towards the presence of additional molecules in the cell except for proteins. Miescher identified the presence of a highly acidic molecule that he isolated from the pus cells and labeled it “nuclein”. The term was coined as the DNA isolated by Miescher was not protein and was derived from the nucleus of the cell. It wasn’t until 1944, when Oswald Avery proposed the DNA as a genetic carrier of information that the Miescher discovery was brought back to light.
Following the X-ray crystallography, by Rosalind Franklin and the determination of DNA double helix by Watson and Crick in 1953, further enhanced the understanding of DNA structure and allowed for the establishment of central dogma of molecular biology. However, one of the flaws with central dogma was the postulation that information flow proceeds from DNA to RNA to protein, which hinders the understanding of different regulatory mechanisms.
In 1955, George Palade identified the first ncRNA as a part of the large ribonucleoprotein complex (RNP). The second class of ncRNA to be discovered was transfer RNA (tRNA) in 1957. However, the first regulatory ncRNA was a microRNA discovered in 1988 from E.coli and was labeled as micF. On other hand, the first eukaryotic microRNA was discovered in C.elegans in 1993. It was derived from gene lin-4 and was identified as a small RNA molecule (as compared to longer mRNA molecules) forming stem-loop structures. This structure gets further modified to generate a shorter RNA that is complementary to the 3’UTR region of lin-14 transcript. This pathway allowed for a better understanding of different post translational gene silencing pathways. Since then, many other miRNAs have been discovered.
Detailed understanding of the mechanism behind this post translational silencing pathway was established in 2001 by Thomas Tuschl. It was discovered that the double stranded RNA gets processed into a shorter 25 nucleotides long fragment which is then modified into a short hairpin like structure by Drosha complex. The molecule is then diced by dicer enzymes into a functional double stranded RNA (dsRNA). These are then loaded onto the RISC complex which then finds and cleaves the targeted mRNA of interest in the cytoplasm.
It wasn’t until 1989 that the imprinting genes were discovered and the genome imprinting was established. The first two genomic imprinting genes were paternally expressed Igf2r and H19. These were both discovered independently in mice and were localized to chromosome 7. H19 is peculiar as it functions as a lncRNA but undergoes modifications similar to that of pre-mRNA processing such as splicing, 3’ polyadenylation and is transcribed by RNA polymerase II. This lncRNA plays a significant role in mice embryonic development and can be lethal if expressed during prenatal stages. More lncRNAs have been discovered in eukaryotes overtime. One such discovery that allowed for better understanding between H19 functions was a lncRNA called XIST (X inactive-specific transcript).
ncRNA drugs and therapy The first ncRNA therapeutic drug approved by food and drug administration (FDA) (1998) and the European medicine agency (EMA) (1999) is called Fomivirsen or Vitravene. The target organ is the eye and works against the cytomegalovirus (CMV) retinitis in immunocompromised patients. The drug functions as an antisense oligonucleotide and binds to the complementary sequence of the mRNA that inhibits the replication of human cytomegalovirus. This therapy can also be categorized as Antisense oligonucleotide (ASO) therapy. There have been many ASO RNA therapeutics that have been approved by FDA and/or EMA over the years, but it wasn’t until 2018 that the EMA approved the drug called Patisiran/Onpattro. The drug uses ds-siRNA as a mechanism of action and is deemed effective against hereditary transthyretin amyloidosis. The mechanism specifically targets the Transthyretin (TTR) mRNA. RNA therapeutic targets are not limited to mature mRNA but have been used to target mRNA at different stages of maturation. One such example is Nusinersen (Spinaraza), it functions as an ASO and targets pre-mRNA before splicing that corresponds to Survival of motor neuron 2 gene (SMN 2). This drug therapy was approved by FDA and EMA in 2016 and 2017 respectively. There are some drugs that have been approved by FDA and not by EMA. This can be seen in the case of an ASO type therapeutics called Eteplirsen (Exondys51) which has been approved by FDA in 2016 but not by EMA. It targets pre-mRNA corresponding to Dystrophin (DMD) and works against Duchenne muscular dystrophy. There are many additional therapeutics that have been developed and are either in phase I or II of the clinical trials. Current RNA therapeutics in clinical trials range from a variety of target organs and diseases ranging from skin (potential treatment for disease such as keloid) to tumors (squamous cell lung cancer).
To date, for both the FDA and the EMA, ncRNAs are considered as "simple" medical products because of their production by chemical synthesis. When some of them, produced biologically (known as bioengineered ncRNA agents: BERAs), will be put on the market, the status of biological medical products will be applied, which could lead to inconsistencies in the legislation.
Applications
Antisense oligonucleotides
Antisense oligonucleotides (ASOs) are single-stranded DNA molecules with full complementarity to one select target mRNA and may act by blocking protein translation (via steric hindrance), causing mRNA degradation (via RNase H-cleavage) or changing pre-mRNA splicing. These short oligonucleotides have already been approved by the FDA for ten genetic disorders and many are currently in the pipeline to be approved/tested. Using oligonucleotide technology, we are now able to control protein expression via RNA interference, and are able to affect previously defined “undruggable” proteins. Even though this therapy has a lot of promise and potential, it comes with many limitations.