The discovery of microRNA opened new doors for drug research. Now, tools are helping researchers study even more regulatory RNA molecules.
In the past few years, researchers have discovered hundreds of small, non–coding RNAs called microRNAs (miRNAs) that are involved in controlling expression of genes throughout eukaryotic genomes. These single-stranded RNA sequences are produced endogenously, are highly conserved in evolution, and their discovery is revolutionizing both basic biomedical research and drug discovery. Unlike the more familiar messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA) molecules that directly mediate protein expression by being involved in amino acid assembly, these miRNAs are not directly involved in protein synthesis. Instead, they control gene expression by interacting with the mRNA molecules themselves, preventing protein synthesis. This means that they do not code for specific proteins, but instead are extremely important in gene expression, emerging as crucial cellular regulators that serve widespread functions in the regulation of cell differentiation, cell proliferation, and cell death.
MicroRNAs function through triggering mRNA degradation or inhibition of protein translation. This posttranslational suppression of gene expression is achieved through binding to the complementary sequence of target mRNA. This binding event causes translational repression of the target gene and also stimulates rapid degradation of the target transcript. There are many gene regulatory factors that control the expression of genomic information, and with more than one-third of human genes thought to be regulated by miRNAs, these molecules represent the greatest number in eukaryotic genomes.
 click to enlarge Figure 1: The mechanism of miRNA transcription, processing, and regulatory activity. MicroRNA genes are transcribed by RNA polymerase II to form primary miRNA (pri-miRNA) molecules. The ribonuclease Drosha then cleaves the pri-miRNA to release the pre-miRNA for cytoplasmic export and processing by Dicer. The mature miRNA product associates with the RNA-induced silencing complex for loading onto the 3’ UTR of target mRNAs to mediate translational repression. (Source: IDT) |
MicroRNA genes are initially transcribed as long RNA precursors (pri-miRNAs), which are then processed to the shorter, hairpin-shaped pre-miRNAs, before they are cleaved to form the mature single-stranded miRNAs. In general, an miRNA is composed of a highly conserved core sequence of 21 to 23 nucleotides (mature miRNA) within a less conserved precursor sequence (pre-miRNA), ranging in size from about 60 to over 120 nucleotides. This pre-miRNA sequence is part of a larger primary transcript that may contain a single pre-miRNA or two or more pre-miRNAs arranged as paired or polycistronic transcripts. Following transcription, pre-miRNAs form a characteristic stem-loop structure that is processed by the RNase III enzyme DROSHA in concert with accessory proteins such as DGCR8 (DiGeorge syndrome Critical Region Gene 8).
The pre-miRNA is then exported from the nucleus to the cytoplasm, where its presence as a double-stranded RNA initiates the RNA interference (RNAi) pathway. Here it is further processed, first by the enzyme DICER, which binds to the hairpin and excises it to a 21 to 23-nt long double-stranded species that is formed by the mature miRNA and a perfect or nearly perfect complementary strand. This RNA has a characteristic structure in which the two strands each protrude at the 3’ end by two nucleotides.
At this point, this small double-stranded RNA is handed off to a protein complex known as RISC (the RNA-induced silencing complex). A protein selects the mature miRNA and “loads” the selected strand onto RISC. It is RISC that transports it to a target mRNA whereupon the mature miRNA binds—usually to the 3’ untranslated region (3’ UTR)—to carry out its regulatory function and either suppresses or completely eliminates translation of that mRNA target. This entire process, whether in animal, plant, or viral genomes, is generically known as post-transcriptional gene silencing (Figure 1).
MicroRNAs put their signatures on disease
Growing evidence shows that miRNAs exhibit a variety of crucial functions related to cell growth, development, and differentiation, controlling the levels of potentially large numbers of proteins. To date over 1500 miRNAs have been identified that regulate the expression of more than one-half of all eukaryotic protein-encoding genes, including human genes.
The involvement of specific miRNAs in mechanisms responsible for switching genes on and off has been shown to modulate distinct altered expression patterns of protein-coding genes. Furthermore, increasing evidence indicates that these expression patterns, among tissues and cells in different differentiation stages, contribute to the initiation and progression of diseases, such as several human cancers, as well as playing a role in regulating cell development, metabolism and viral infections. The mis-expression of miRNAs has also been shown to be necessary and sufficient for multiple forms of heart disease. Hence evaluation of the global expression of miRNAs potentially provides opportunities to identify regulatory points for many different biological processes, and can therefore be used to monitor different cell states.
MicroRNAs are making headlines in drug discovery for their ability to fine tune the activity of genes that play critical roles in various biological pathways. Research has been intensely focused on studying and understanding miRNA expression for investigating novel miRNA-based therapeutic opportunities, with both the miRNA itself and its regulatory target being important potential drug targets because their malfunction has been implicated in several disease states. MicroRNA expression profiles have shown to be particularly effective for classifying human cancers and have already shown great potential for early cancer detection and successful treatment before cells metastasize. As many cancers are accompanied by low levels of miRNA, the administration of metabolically-stabilized miRNA analogues as a cancer therapy is currently being explored. Conversely, where diseases are characterized by increased levels of particular miRNAs, the inhibition of their function may be preferable. Understanding how this works has the potential to deliver a range of new therapies as biological markers covering multiple disease categories, including patient prognosis and treatment evaluation.
 click to enlarge Figure 2: The miRCat cloning method. Both the small RNA enrichment and 3’ ligation purifications are carried out on 12% denaturing (7M urea) PAGE gels. Concatamerization is based upon the SAGE (serial analysis of gene expression) technology and is presented as an alternative to “shot-gun” PCR cloning. (Source: IDT miRCat microRNA Cloning Kit Technical Manual) |
Detecting miRNAs
MicroRNAs are one of six (as yet) known classes of small regulatory RNAs and are the only class that form stem-loop hairpin structures. This means that potential miRNAs can be predicted on the basis of conserved hairpins using genomic sequences. However, validation of predicted miRNAs and discovery of the other small regulatory RNAs that do not form hairpins can only be done by direct cloning and sequencing. There are various methods of visualizing small RNA molecules, such as Northern blots, quantitative PCR, and microarrays, but while these allow expression profiling they can only be used to detect molecules that are already known.
As researchers continue to study miRNA expression in diseases and focus on the most relevant miRNAs, further advancement in miRNA detection technologies and bioinformatics algorithms is crucial for success. In concert with this, identification of new small RNA molecules—a more complicated protocol involving enriching, amplifying, cloning, and sequencing—must keep pace. This highlights the importance of developing techniques for finding new regulatory RNA molecules that are being shown to play such an important part in disease onset and progression, as well as possibly being useful as therapeutic candidates.
A generic protocol for cloning and sequencing small RNAs was developed by Integrated DNA Technologies (IDT) and commercialized as the miRCat small RNA cloning kit.
It enables the creation of small RNA (including miRNAs, piRNAs and endogenous siRNAs) libraries from any primary RNA source and accounts for the natural variability in structure and sequence between species.
Based upon a pre-activated, adenylated oligonucleotide linkering method, it consists of three sequential protocols: RNA isolation and enrichment, followed by cloning linker attachment, and ending with amplification and a cloning phase.
in vitro miRNA cloning
Employing the miRCat small RNA cloning system, IDT directly cloned and identified conserved and nonconserved miRNAs in the genome of the gray short-tailed opossum Monodelphis domestica. This small South American species is the world’s most widely used marsupial model for biomedical research and is the first marsupial genome to be sequenced [Devor and Samollow, 2008; Devor et al., 2008].
MicroRNA cloning was carried out against an RNA pool composed of five major organs—brain, heart, lung, liver and kidney—from an adult female marsupial. Mature sequences were successfully cloned for the majority of miRNAs conserved among mammals. Among these sequences were several that occurred in many of the clones. One of these, miR-122a, is a liver-specific miRNA associated with hepatitis C viral replications. Another sequence, miR-29b, has been shown to control expression of a specific branched amino acid complex [Devor and Samollow, 2008]. In addition to validating evolutionarily conserved microRNAs, 15 new, marsupial-specific miRNAs and more than 300 members of another class of small RNA called PIWI-interacting RNAs (piRNAs) have been found using the miRCat protocol [Devor and Samollow, 2008; Devor et al., 2008].
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Conclusion
This technique can be used to identify miRNAs and other classes of small, regulatory RNAs. Compiling miRNA profiles in any animal or human is crucial for the developmental roles being assigned to specific miRNAs. Moreover, the discovery of these miRNAs will also shed light on miRNA evolution, as well as on the possible processes they are regulating.
MicroRNA research holds great promise for the future. These natural gene regulation agents may be the backbone of many of the most cutting-edge medical therapies and provide an effective new avenue of drug discovery.
About the Author
Devor received his PhD in Population Genetics from the University of New Mexico in 1979. He joined IDT’s Molecular Genetics Research Group in 1998, and has more than 80 scientific publications and three US patents in molecular genetics methods..
This article was published in Drug Discovery & Development magazine: Vol. 10, No. 9, September, 2008, pp. 26-30.
Filed Under: Genomics/Proteomics
