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Encyclopedia of Cancer, 2015
Noncoding RNAs are RNA molecules that do not encode proteins. They constitute the vast majority of human genome, ~97 % (Fig. 1). Recent research revealed functional roles for several noncoding RNAs, but it is not known if all are functional.
Types of noncoding RNAs
Several types of noncoding RNAs are now well characterized as shown in Fig. 2 and explained below. For further reading, the reader is referred to recent review article (Esteller 2011).
Transfer RNA (tRNA)
tRNA molecules are 73–93 nucleotide long which transfer amino acids to the translation site of the messenger RNA (mRNA) according to the genetic code during protein synthesis. The secondary structure of tRNA is usually visualized as cloverleaf and the tertiary structure is L-shaped.
Ribosomal RNA (rRNA)
While tRNA is responsible for amino acid transfer to the site of protein synthesis, rRNA is part of the machinery (ribosome) through which the mRNA is decoded into amino acids. rRNA consists of two subunits, a large subunit and a small subunit. These subunits catalyze the formation of peptide bonds that link amino acids during protein synthesis.
Small nuclear RNA (snRNA)
snRNAs are localized in the nucleus and divided into small nucleolar RNAs (snoRNA) such as U3 RNA and small Cajal body RNA (scaRNA) such as U85. snRNAs are involved in several cellular processes such as transcription and RNA splicing.
RNA interference (RNAi)
These are small double-stranded RNA molecules that target specific RNA sequence resulting in gene silencing. They include small interfering RNA (siRNA), repeat-associated small interfering RNA (rasiRNA), piwi-interacting RNA (piRNA), and microRNA (miRNA).
Fig. 1. Adapted from http://genomicenterprise.com/non-coding_rnas. The percentage of noncoding sequences increases with organisms complexity
Fig. 2. Types and classes of noncoding RNAs. RNAs are classified to coding RNAs and noncoding RNAs (Fig. 2). As the name suggests coding RNAs encode for proteins whereas noncoding RNAs do not. They are divided to transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), interference RNA (RNAi), and long noncoding RNA (lncRNA). snRNAs are subdivided to small nucleolar RNA (snoRNA) and small Cajal body RNA (scaRNA). The RNAi group is subdivided to small interference RNA (siRNA), piwi-interacting RNA (piRNA), repeat-associated small interfering RNA (rasiRNA), and microRNA (miRNA)
These double-stranded RNA molecules are very effective in gene silencing. Initially siRNA was discovered in plants and then in Caenorhabditis elegans worm and mammals. For siRNA to silence gene expression, the double-stranded RNA is processed by a group of enzymes including RNase III and Dicer and finally loaded onto the RNA by a complex called the RNA-induced silencing complex (RISC). RNAs bound to RISC are directed to degradation by endonucleases and exonucleases. The RNAi technology is widely used to downregulate specific genes assisting researchers to understand the functions of these genes.
MicroRNAs are a large family of small RNAs. They are about 22 nucleotides long. The biogenesis is meditated by a group of enzymes including Drosha and Dicer (Fig. 3). For further reading, the reader is referred to review article (Bartel 2004). Like siRNAs, miRNAs are loaded onto the target transcript by RISC. miRNA molecules also silence gene expression by enhancing messenger RNA (mRNA) degradation or suppressing its translation. In plants, miRNA sequences complement the target sequence almost perfectly. On the other hand, in mammalian cells, miRNA sequences mainly complement a seed region of at least six bases. MicroRNAs normally target the three prime untranslated regions (30UTR) of the mRNA. However, it can also target a sequence in the 50UTR or coding region (CR). The first miRNAs were identified in the early 1990s, and now more than a thousand miRNAs are identified. Some miRNAs are known to be tissue specific such as microRNA-1 which is mostly expressed in muscle and heart. They are also differentially expressed in cancer versus normal cells. For example, microRNA-148b is frequently downregulated in gastric cancer, while miR-155 is highly expressed in many human cancers including breast cancer. MicroRNAs are also influenced by stresses such as cigarette smoke that causes a downregulation of microRNA expression levels in rat lungs.
Fig. 3. Illustration of microRNA (miRNA) synthesis pathway
These are small noncoding regulatory RNAs which are also involved in gene silencing as microRNAs and siRNAs. However, they differ in length and can be 27 nucleotides long. These RNAs interact with piwi subfamily proteins and thus classified as piwi-interacting RNAs (piRNAs). They were first discovered in Drosophila melanogaster, and later similar RNAs were identified in mammalian cells. RNA profiling studies in the fiy revealed that rasiRNAs are associated with proteins known as Argonaut 3 (Ago3), Aubergine (Aub), as well as piwi proteins. The process of biogenesis of these RNAs is still not well understood. However, scientists proposed a model known as “ping-pong.” In this model, rasiRNA guides piwi protein complex to cleave the 50 end of the target RNA and that generates the new rasiRNA. Studies in Drosophila melanogaster revealed that two nuclease enzymes (zucchini and squash) are involved in this process. The 30 end of rasiRNAs were found to be 20-O-methylated and that may dictate the site of cleavage.
Long noncoding RNAs (lncRNAs)
lncRNAs are also known as long intergenic noncoding RNAs and recently also called long intervening noncoding RNAs (lincRNA). These RNAs are more heterogeneous than small RNAs. They vary in length from few hundreds to thousands of nucleotides. They are involved in several processes such as epigenetic modifications, chromatin accessibility, and X-chromosome inactivation. They constitute the largest RNA group of about 6,000. lncRNAs are transcribed from the intergenic regions; however, their processing is not clearly understood. Unlike microRNAs and siRNAs, lincRNAs can modulate gene expression both positively and negatively, and they can regulate the expression of neighboring genes as well as distant genomic sequences. For example, they can mediate gene activation by serving as molecular signals and suppress gene expression by acting as a decoy RNAs that can sponge transcription factors (essential for gene expression). They can also act as molecular guides by recruiting RNA-protein complexes to chromatin and thus affecting gene expression of neighboring genes or even distant genes. Some lincRNAs can act as a scaffold in a complex that may influence transcriptional activation or repression.
Noncoding RNAs and cancer
snoRNAs are differentially expressed in non-small cell lung cancer compared to the normal matched tissue. Prostate cancer development and breast cancer were found to be associated with the deletion of the snoRNA U50. snoRNA-associated proteins may also be linked to tumorigenesis by influencing ribosomes and protein translation, which is frequently disrupted in cancer cells.
microRNAs are the most studied noncoding RNA with respect to cancer. Researchers have profiled microRNA expression in several human cancer tissues and compared with normal tissues. While the majority of microRNAs are downregulated in cancer cells, some are upregulated. Researchers also found that they can suppress tumorigenesis (known as tumor suppressor microRNAs) or enhance tumor progression (known as onco-microRNAs). One of the first examples was microRNA-15 and microRNA-16 which are dysregulated in B-cell chronic lymphocytic leukemias. In addition, microRNAs are quite often located in fragile chromosomal regions linked to cancer such as breast, ovarian, and melanoma. Dysregulation of microRNA in cancer may arise from defects in microRNA processing machinery such as mutation of Dicer in familial pleuropulmonary blastoma. Defects in Dicer and other proteins involved in microRNA processing contribute to clarifying the defects in microRNA expression in cancer cells.
piRNAs are also involved in tumorigenesis such as testicular tissue cancer. The role of these noncoding RNAs in tumorigenesis is unknown. However, piwi proteins (associated with piRNAs) are highly expressed in different somatic tumors, linked to malignant differentiation and drug resistance. Studies on Drosophila melanogaster suggest that piRNA machinery may promote brain tumor growth.
lncRNAs are altered in different types of human cancers. For example, lncRNA HOTAIR is involved in human neoplasia and is the most well-understood lncRNA. Cancer cells invasiveness increases when HOTAIR expression is low. It is possible that HOTAIR may be playing a role in mediating cell transformation. Other lncRNAs such as lincRNA-p21 are postulated to have similar function. This cannot be generalized since we still do not know the functions of most lncRNA (Esteller 2011).
Noncoding RNAs and therapy
Noncoding RNAs provide a new therapeutic approach as well as those proteins involved in their biogenesis. So far, miroRNAs are most investigated in the context of cancer therapy. For example, in an approach to inhibit a single microRNA, in mouse mammary tumor model, inhibition of microRNA10b was found to prevent metastasis. In another approach, multiple microRNAs were simultaneously inhibited. For example, microRNAs 21, 155, and 17-5p were inhibited in several tumors. This approach resulted in more inhibition of cancer cell growth compared to individual microRNAs. Similar approach can be designed to control other process in cancer such as angiogenesis, invasion, and metastasis.
The above strategies are aimed to microRNA inhibition; however, restoration of microRNA levels in cancer cells can be an alternative strategy. This approach is known as “microRNA replacement therapy” as researchers re-expressed microRNA-26a in hepatocellular carcinoma. This resulted in suppression of cancer cell proliferation in mouse hepatocellular carcinoma. As mentioned above, most microRNAs are downregulated in cancer cells. Thus, similar approach to restore microRNA levels in cancer cells was developed (known as microRNAome). It globally restores microRNA expression in cancer cells and thus prevents tumor growth resulting in cell death. This promising approach received approval for further possible clinical use to treat certain malignancies.
It is believed that other noncoding RNAs and, when discovered, the unknown processing machinery will also receive a considerable interest as microRNAs. It is challenging to inhibit a long noncoding RNA with the same approach as microRNAs. However, siRNAs may be used to achieve lncRNA inhibition.
Finally as Thomas R. Gingeras elegantly said, “We should not be too sceptical about non-coding RNAs just because we don’t know their functions”(Kowalczyk et al. 2012).
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297 Esteller M (2011) Non-coding RNAs in human disease. Nat Rev Genet 12(12):861–874. doi:10.1038/nrg3074, PMID: 22094949
Kowalczyk MS, Higgs DR, Gingeras TR (2012) Molecular biology: RNA discrimination. Nature 482(7385):310–311. doi:10.1038/482310a