Alltech, a Kentucky-based company aiming to improve agricultural sustainability via biotechnologies, hosts a competition each year called the Alltech Young Scientist competition, for both the undergraduate and graduate levels. Submissions can be original research or original review papers. In 2013, I submitted a review paper explaining & exploring “microRNA” and, of the >5500 entries across North & South America, I ended up placing 3rd!
With everything going on at the time, I never pursued publication, and although it is pretty old now, I think there are some solid concepts outlined for anyone unfamiliar with microRNA. I hope to revisit this topic and update and/or add newer concepts, but for now, here it is verbatim in all its technical, jargon-laden glory:
MicroRNA: Tiny Transcript, Huge Impact
MicroRNA (miRNA) is a recently-distinguished eukaryotic class of very small non-protein-encoding RNA transcripts that interact with messenger RNA (mRNA) molecules to act as a double-stranded post-transcriptional regulatory mechanism, either by gene silencing from its catalytic initiation of mRNA degradation, or through translational repression via binding with mRNA. These previously-unnoticed transcripts are generated as the product of miRNA-encoding genes in seemingly random locations of the genome, and undergo several stages of processing, to finally interact as a single-stranded (ss) RNA transcript with several proteins of an RNA-induced silencing complex (RISC) which, as a whole, demonstrates post-transcriptional regulation.
Due to the small size of the miRNAs, many new modifications to conventional analyses have been developed. The assessments made possible by these novel techniques have increased accuracy in quantitating and characterizing miRNAs, providing information about miRNAs that is increasingly conveying their fundamental importance, and also escalating attention towards a new area of research. Indispensable miRNA influences on biological and metabolic processes, such as growth, development and its phase changes, damage recovery, and stress-response mechanisms – in both plants and animals – have been revealed. What is more, compatibility across kingdoms has been observed. As such, numerous methods for profiling of unique miRNA expression patterns have been developed.
miRNA studies are continually providing insight on their significance in regulating genetic expression, albeit sometimes complex, suggesting (and recently, demonstrating) many clever implementations in disciplines such as genetic sequencing, clinical disease diagnoses, histopathology, neurology, reverse genetics, and genetic predictors. These ideas, sparked by the unique molecule that is miRNA, have begun an exciting direction of research, which will be applicable to humans and mammals, plants, viruses, and fungi. miRNAs offer alternatives to conventional medicine, breeding, and controversial genetic techniques.
Introduction & History
Over thirty years ago, while studying bacterial plasmid maintenance, Tomizawa et al. noticed that DNA transcription by an RNA polymerase started at two sites within a required plasmid maintenance region (1981). A transcript produced at one site, after being cleaved by a ribonuclease (RNase), became primers for DNA replication, while the second site produced a short RNA molecule (Tomizawa et al., 1981). It was observed that, when purified, this short RNA (roughly 100 nucleotides (nt) in length) would inhibit formation of the primer acting on that template whence it came, and only on this template. Preformed precursors showed normal activity, thus Tomizawa et al. concluded that this highly-specific translation primer inhibition was dependent upon the hybrid formation between the short RNA primer precursor, and a template (1981).
Shortly after, while trying to discern more effective methods of identifying and characterizing genes that regulate and contribute to fundamental cellular and embryological processes at the Fred Hutchinson Cancer Research Center, Izant and Weintraub investigated the precedent notion of an antisense DNA strand inhibiting gene activity (1984). At the time, genetic contribution to a physiological or developmental stage was studied by using metazoans as a model organism. Although an excellent model, techniques of the time often required induced mutations to their genes, consequently damaging and even destroying the organism; to compensate for this, drugs or antibodies were often introduced to living cells as specific inhibitors, adding needless complexity and time to an already-damaging process (Izant & Weintraub, 1984). Therefore, by cotransforming mouse cells with a reverse-oriented herpes simplex virus thymidine kinase (TK) gene, Izant and Weintraub investigated an alternative to using mutating agents to interact with a gene product by “investigating whether nucleic acid sequences, complementary to that of a predetermined mRNA, can inhibit the expression of that gene by RNA duplex formation” (1984). Their results suggested that the “anti-message” was due to a trans-inhibition of TK causing a reduced capacity for its expression (Izant & Weintraub, 1984).
Several years later, a new mitochondrial, small RNA, “guideRNA” (gRNA), was hypothesized to have a role in RNA editing (Blum et al., 1990). Encoded by intergenic segments of DNA, gRNAs demonstrated highly-specific complementarity (of about 50nts) with mature mRNAs of edited regions, with the 5’ end having the ability to partially hybridize with the 3’ end of the edited mRNA region, and allow full hybridization between the gRNA and the mature mRNA. In the same year, one group remarked that a trans-regulated mouse gene of ~2.6Kb in size did not encode for a protein, although 35 putative open reading frames (ORFs) were examined; it was proposed that this gene encodes for several small RNA molecules (Brannan et al., 1990). Another study suggested that small lengths of nucleic RNAs may be involved in RNA processing, attempting to add to the discernible information of intron function in processing pre-mRNAs (Guthrie, 1991).
In 1993, while examining the temporal fates of cells throughout larval development in C. elegans, it was observed that the gene lin-4 was temporally essential for specific larval development stages via its inhibitory effect on lin-14 (and thus, on its product, LIN-14) (Lee et al., 1993). Successful transformational rescue of lin-4 loss-of-function mutants of several species provided evidence for the inhibitory effect of lin-4 on translation, and additional sequencing revealed the highly conserved nature of the sequence (Lee et al., 1993). Interestingly, lin-4 acted as a post-transcriptional regulator not by producing a protein, but by encoding for very small (22 and 61nt) RNAs, with antisense complementarity to the 3’ untranslated region (UTR) of lin-14. An indication of evolutionary requirement of miRNAs was sparked when high conservation across species was observed. Furthermore, it was predicted that these small RNAs could fold into stem-loop or hairpin structures, once active, to act as a regulatory mechanism (Lee et al., 1993). This would later be considered by many to be the first miRNA discovered. Half a decade later, and building on these results, Fire et al. determined that interference caused by small RNAs was exceptionally higher when introduced as a dsRNA molecule – even with as little as a few molecules per cell (1998).
The second miRNA, let-7, was also confirmed in C. elegans not long after lin-4 (Reinhart et al., 2000). It was concluded that let-7, much like lin-4, is a heterochronic switch gene with complementarity to a portion of an mRNA 3’ UTR. let-7 produced 21nt RNAs, not proteins, suggested to directly influence necessary precursors for various functional proteins needed at each developmental stage. By indirectly triggering development stage transitions through interaction with templates of heterochronic protein complements specifically involved with the regulation of developmental stages, it was proposed that this mechanism elicits a cascade of developmental, stage-specific RNA transcription (Reinhart et al., 2000). Remarkably, let-7 RNAs were also discovered in ascidians, mollusks, annelids, and arthropods, shaping a more confident perception of biological and evolutionary significance (Pasquinelli et al., 2000).
Clearly, these small RNAs were becoming the focus of one intriguing novel research field. Adopting one common designation for the tiny molecules was inevitable. The conventionally-used term “microRNA” was designated concurrently by three groups in 2001 (Lau et al., Lee & Ambros, and Lagos-Quintana et al.). Additionally, they determined a multitude of new miRNAs, their genes, and predicted secondary structures in C. elegans, and offered much insight into the diversity and variability of miRNAs. In fact, Ambros et al. initiated a new system for identifying and annotating miRNAs to maintain consistency while upholding unpublished confidentiality, to set them apart from other similar small RNAs, and to establish an online directory (2003).
Pertaining to human health, one of the first studies linking miRNAs to pathology was in 2002 (Calin et al.). It was observed that two specific miRNAs had been deleted at high frequency in humans with chronic lymphocytic leukemia; these miRNAs were physically located near one another on the chromosome, and in 68% of cases studied, these miRNAs were down-regulated or omitted (Calin et al., 2002).
In the years to follow, miRNAs were identified in an increasing number of tissue types, gene families, and developmental stages (Lim et al., 2003). Furthermore, a variety of organisms and physiological conditions were linked with specific expression patterns of miRNAs, such as flies and worms (Ambros, 2003), plants (Lagos-Quintana, 2001; Lau et al., 2001; Lee & Ambros, 2001, Reinhart et al., 2002), mammals (Lewis et al., 2003; Rodriguez et al., 2004), viruses (Pan et al., 2007), and fungi (Lee et al., 2010) as well as human physiological conditions such as cardiac hypertension (Thum et al., 2007), cardiac arrhythmogenesis (Yang et al, 2007), regulation of circadian rhythm (Xu et al., 2007), cancer tissue growth (Rosenfeld et al., 2008) and metastasis (Ma et al., 2007; Tavazoie et al., 2008), and even aging (Zhang, 2011; Smith-Vikos & Slack, 2012). The recent better understanding of miRNAs and their functional mechanisms have led to the use of exogenous and synthetic miRNAs to affect regulating mechanisms directly involved with cancer tumor suppression in mammals (Chen et al., 2012). Evidence of genetic conservation has contributed to the widely-accepted view that miRNAs are a genetic regulating mechanisms conserved for over 400 million years (Floyd & Bowman, 2004). Today, Zhang precisely defines miRNA as “…small RNA molecules (~22 nucleotides) that regulate gene functions either via degradation of mRNAs that form perfect match between miRNAs and their cognate mRNAs, or by inhibition of protein translation via imperfect match with the 3’UTR…of the mRNA transcripts” (2011).
miRNA Biogenesis
miRNA-encoding genes are found in the organisms’ chromosomes, either individually, as often observed in Drosophila (Lim et al., 2003) or as clusters with other functionally-related miRNA-encoding genes (Aravin et al., 2003), consequently sharing one promoter as a polycistronic expression system (Figure 1). Some miRNA-encoding genes may be unique genetic portions, while others may be included as part of protein-encoding genes (Wang & Yang, 2010). Wang & Yang designate two classifications for miRNA genetic origin: intergenic, in which miRNA-encoding genes are found between protein coding genes (~42% of total miRNA genes in humans), and intragenic, those within a host gene (~48% of total) (2010). Intragenic miRNA-encoding genes are further classified as intronic, exonic, 5’UTR-originating, or 3’UTR-originating.
For some time, it was believed that miRNA gene transcription was due solely to RNA polymerase II since its direct involvement was confirmed in 2004, due to the unique cap structures and poly-adenosine(A) tails added to the product, unique to that class of polymerase (Lee et al., 2004). However, Borchert et al. confirmed the transcription of at least 50 mammalian miRNAs was due to the activity of RNA polymerase III (2006).
The products of transcribed miRNA genes are precursors to mature miRNAs, labeled “primary-miRNA” (pri-miRNA) (Lee et al., 2002). These single-stranded precursors can be several kilobase (kb) pairs long, and can be derived from genes containing protein-encoding exons, non-coding introns, as well as poly- or mono-cistronic miRNA genes as seen in Figure 2 (Ying & Lin, 2005; Lee et al., 2002). The pri-miRNA transcripts have perfect or near-perfect complementarity, often with a location physically near to that whence it originates, thus they form a secondary stem-loop or hairpin structure, followed by entry into the miRNA-processing pathway.
At this stage, there are two methods of entry, defined by the ribonuclease (RNAse) endonuclease-III (DROSHA) activity (or lack thereof), thus being either DROSHA:DROSHA-mediated, or DROSHA:DROSHA-independent (Ruby et al., 2007). The DROSHA-mediated pathway requires the co-activity of DGCR8, a protein which helps DROSHA recognize the single-strand/double-strand junction at the base of the pri-miRNA stem-loop or hairpin. Once activated, DROSHA cleaves the base of the stem or hairpin 11nts from the single stranded segments to liberate a smaller dsRNA segment, ~70nt in length and with the loop intact, but resulting in a staggered, 2nt overhang on the 3’ end of the dsRNA opposite the loop, now designated “precursor miRNA” (pre-miRNA) (Lee et al., 2002).
The alternative method of entry to the miRNA-processing pathway, and much less common, is DROSHA-independent (Ruby et al., 2007). This pathway occurs when debranched introns mimicking structural features of pre-miRNAs are spliced from the pri-miRNA transcript to form a pre-miRNA, similar to the product of the first pathway (Ruby et al., 2007; Renalier, 2011).
In animals, the pre-miRNAs are then actively transported from the nucleus to the cytoplasm via Ran-GTP and Exportin-5 (Yi et al., 2003). Here, they undergo a second double-stranded cleavage via Dicer, a cytoplasmic RNase III endonuclease which recognizes the 5’ phosphate and 3’ 2nt overhang, producing the mature miRNA duplex (Bartel, 2004). The mature duplex is composed of miRNA, the “guide strand,” and miRNA*, the “passenger strand” (Lau et al., 2001).
In plants, the mature duplex is formed in the cytoplasm before being transported to the cytoplasm (Reinhart et al., 2002). The loop formation is removed by cleavage activity of the nuclear Dicer-like enzyme 1 to produce the miRNA duplex (Lee et al., 2002b). It has been proposed that HASTY, an ortholog to animal Exportin-5, transports the duplex into the cytoplasm (Bartel, 2004).
It was demonstrated that this mature duplex nature is fundamental in attributing functionality to miRNA, requiring only several strands per cell to display an effect (Fire et al., 1998). They suggested that highly specific and powerful interference is caused by the double-stranded interaction between small RNAs and mRNAs, and their findings suggest that the duality of the strands must have some interactive feature with necessitated regulatory mechanisms.
As the mature duplex miRNA/miRNA*, the structure is still not a functional regulator of expression; the duplex must separate to single-strands, where it can interact with an RNA-induced silencing complex (RISC) to become a functional post-transcriptional repressor, called “miRISC” (Martinez et al., 2002). RISC contains several regulatory proteins, such as the PAZ domain, which interacts with the 3’ end of the soon-to-be active miRNA, as well as the N-terminal and middle domains which complete the entirety of RISC and help it conform to proper binding structures in its target (Wang & Yang, 2010). Moreover, various Argonaute (Ago) proteins may be found within RISC which exhibit endonuclolytic activity within its Piwi domain (Renalier, 2011). Thus, miRNAs are not accurately the translational repressor; rather, their association with and guidance of a greater protein complex as a whole is the post-transcriptional regulator. A cytoplasmic helicase splits the duplex into individual strands; the two strands of the duplex are highly similar, but there is a slight asymmetry at the duplex extremities with which they are paired (Schwarz et al., 2003). This slight difference in pairing strength allows RISC to selectively distinguish between the two strands. Normally, the phosphate group of the strand with the less-stable 5’ end will bind more strongly with RISC, thus becoming the miRNA guide strand (Schwarz et al., 2003). miRNA* is hence degraded. Although the single-strand that binds with RISC is often the guide strand, there have been instances in mouse tissue where both strands were equally accumulated (Ro et al., 2007) and in Drosophila where the miRNA* has bound with the complex to direct the translational repression (Renalier, 2011). Maybe more beguiling, there have been observations in differences in frequency of tissue-dependent binding of the two strands from one duplex within a single organism, suggesting additional regulatory factors may be involved (Renalier, 2011).
Modes of Action: Structure, Function, Mechanism
Although the majority of miRNAs to date target 3’ UTRs of mRNAs (Renalier, 2011), particularly in animals, miRNA-mRNA target interactions in the 5’ UTR (Sunkar & Zhu, 2004) and coding sequence (He & Hannon, 2004) have also been observed. Studies have found the majority of target sequences in plants to be in the open reading frame (ORF) (Jones-Rhoades & Bartel, 2004) or in humans, present in relatively high numbers within the ORF (Lewis et al., 2005).
As Renalier explains, the 3’ end of the now-active single-strand miRNA contributes stability between RISC and the miRNA, while at the 5’ end, 2-8nts (inclusively) form the “seed sequence,” which acts as the discriminating portion of the miRISC (2011). Moreover, Lewis et al. specify that seed sequences are typically characterized by their flanking adenosine residues (2005). The miRISC targets mRNA to post-transcriptionally regulate expression in two ways. One method is via the endonucleolytic activity, which cleaves target mRNA (although in 2005, Bagga et al. have observed some exonucleolytic activity); in order for this to occur, the miRNA must form a perfect match with its target at the scissile phosphate bond between the 10th and 11th nt of the miRNA. Another requirement is that Ago proteins must have a competent site, and to date, only Ago2 is shown to be effective. Finally, other co-factors may be required to enable cleavage rather than repression. miRISC degradation of its target is initiated by the deadenylation and decapping of the target mRNA (Wang & Yang, 2010). The miRISC can degrade multiple targets, since it remains intact and functional for some time after its activities initiate (Bartel, 2004). As the presence of intact mRNAs decreases, so do their products; in this case, translation rate is constant but there are few transcripts translated. Degradation will occur when miRNAs recognize target sequences in the UTR or coding sequence of the target mRNA (Wang & Yang, 2010).
Renalier purports an alternative to degradation of a target is via translational repression, which diminishes the target’s stability and consequently reduces the translational potential of a target mRNA. This occurs when only seed site-mRNA complementarity exists, and the endonucleolytic activity is not present (Wang & Yang, 2010). It has been suggested that repression of translation is due to either miRISC binding to the target mRNA and thus physically slowing or blocking translation once bound with ribosomal RNA (rather than the mRNA) and thus reducing efficiency of translation, or by blocking the efficient formation of translational machinery on the mRNA transcript (Bartel, 2004; Aukerman et al, 2003). Initially, this was thought to be the sole mechanism by which miRNAs regulate expression, as target transcripts were constant but their expression diminished with increasing miRNA presence (Lee et al., 1993). In contrast to degradation, translation rate is reduced while transcripts remain constant. Wang and Yang indicate that repression, and not degradation, usually occurs when the guiding strand of miRISC is complementary to the 3’ UTR (2010).
Intriguingly, although the vast majority of miRNAs have been shown to down-regulate or silence gene expression (Renalier, 2011), rare cases have proven to be opposite. For example, Vasudevan et al. found that under certain conditions, target mRNAs are transformed into translation activation signals, causing specific miRNAs to direct translation proteins to this site (2007). Additionally, Ørom et al. discovered that a specific miRNA was functionally related to a specific 5’UTR motif of a ribosome-encoding mRNA, and, once bound together, actually enhanced ribosomal translation by altering translation-repressors (2008). In contrast, the same miRNA exhibited some duality in function as it was shown to repress translation when bound to a second mRNA target in the 3’ UTR region (Ørom et al., 2008).
miRNA Expression
Expression of miRNA is described as spatiotemporal (Wang &Yang, 2010). Different tissues, organs, development stages, health conditions have a noticeable effect in miRNA expression levels, while other miRNAs are ubiquitously expressed with regards to locale and age (Lee et al., 1993; Calin et al., 2002; Ambros, 2003; Lim et al., 2003; Liang et al., 2007; Tavazoie et al., 2008). miRNAs are highly variable in level of expression, some are tissue-specific, and some are preferentially-expressed and tissue-dependent (Liang et al., 2007). Chromosomal location of miRNA-encoding genes are typically at fragile, splice, or aberration points in the genome (Wang & Yang, 2010). Liang et al. uncovered 15 unique human miRNAs with relatively constant expression levels in all tissues and life cycles (2007). The original interference RNA (RNAi) was described as the “introduction of RNA into cells [that] can be used in certain biological systems to interfere with the function of an endogenous gene” (Fire et al., 1998). Thus, miRNA is encompassed with this definition (Figure 3).
There has been some correlation observed between expression levels of miRNAs, and the host genes in which the intragenically-oriented genes are found; in fact, 77% of human miRNA expression patterns studied positively correlated with their host gene expression, and both expression levels and relative numbers of miRNA-encoding genes were higher in placental tissue (Wang & Yang, 2010). Studies have led Wang & Yang to estimate that over 30% of protein-encoding genes in humans are regulated by miRNAs (2010). The relationship between miRNA expression is complex; to date, it is not clear whether one miRNA consistently has multiple targets (Zhang, 2009), or if multiple miRNAs can or must regulate a single mRNA.
miRNA and mRNA Target Prediction
Before miRNA expression can be detected, identified, and exploited to its full potential, it is necessary to have the capacity to predict miRNA structure, and therefore its target. This understanding enables an efficient and specific direction of analysis. Due to the potentially vast range of miRNAs, their small size, poorly understood mechanisms, and relatively recent discovery, various bioinformatics sites and engines with unique algorithms have played a major role in their prediction (John et al., 2004; Kiriakidou et al., 2004; Lewis et al., 2005).
Lewis et al. developed an online search method, TargetScan.org, which searches mammalian query miRNA sequences (2005). Its approach utilizes the 8mer and 7mer seed sequence sites to detect conservation, while predicting non-conserved regions; furthermore, it takes into account whole-genome alignments when searching human sequences (Whitehead Institute of Biomedical Research, 2011). miRNAs can be searched by specie name, entrez gene symbol, and with various options to narrow searches such as within families and level of conservation, to produce miscellaneous predictions of sequences and their targets.
Another online method is miRanda at mirbase.org (Memorial Sloan-Kettering Cancer Center, 2010). This method was developed by John et al. by optimizing sequence complementarity (but not strictly seed sequence) of position-specific rules based on strict parameters of interspecies conservation (2004). The database can be searched within several organisms’ genomes, by miRNA/mRNA/tissue-specific criteria to predict sequence and secondary structure, targets, sequences of high homology, and provides links to any related literature.
A third online method, called Diana Lab, was developed in 2004 by Kiriakidou et al. This database provides information that “range from the analysis of expression regulation from deep sequencing data, the annotation of miRNA regulatory elements and targets to the interpretation of the role of miRNAs in various diseases” (Alexander Fleming Biomedical Sciences Research Center, 2012). This site requires a download although it is web based, and searches are based on thermodynamic profiles of each miRNA in addition to statistics and conservation (exiqon, 2012).
Limitless combinations of bioinformatic databases and algorithms can be used effectively. For example, Smallheiser incorporated these bioinformatics methods with a database of expressed sequence tags (ESTs) in GenBank databases to predict animal miRNA mRNA-precursors (2003). Similarly, several new plant miRNAs were identified and characterized using GenBank’s expressed sequence tag (EST) databases in previously-established sequences (Zhang et al., 2005). Bentwich et al. provide an excellent example of a combination of bioinformatic searches with modified high-throughput microarray, PCR, and sequence-directed cloning to discover nearly 100 novel human miRNAs (2008).
None of these predictions are perfect, however. It is important to note that, due to the first miRNAs’ characteristics and presence in the 3’UTR, there has been search bias within those regions in early stages and when these tools were developed. As well, searches may be limited to organisms having had the entire genome sequenced.
miRNA Detection
Although over thirty methods for detecting miRNAs exist (Wang & Yang, 2010), many are slight variations on preexisting methods. These are necessary largely due to the small size of the miRNAs. Because of the potentially high numbers of miRNAs, an accurate yet high-throughput method is ideal; thus, although the Northern blot is the standard for miRNA expression profiling, it is too time-consuming and not feasible for large-scale profiling even after modification for miRNAs (Válóczi et al., 2004). Realistically, there are two main effective methods: optical, and the less-popular electrical (Wang & Yang). Of the widely-used optical methods, there are principally two approaches: using modified oligo probes, and cloning (Vester & Wengel, 2004; Chen et al., 2005; Raymond et al., 2005; Liang et al., 2007; Saba et al., 2008). Electrically, most methods are variations on one technique (Park et al., 2002; Macanovic et al., 2004).
An essential modification for any oligo probe-based technique was the development of locked nucleic acids (LNAs) for microarrays (Vester & Wengel, 2004). LNAs are analogues to nucleic acids, which contain an O2’C4’-methylene-bridge bicyclic furanose unit locked in a conformation mimicking a RNA (Figure 4). These high-affinity analogues are compatible with ds- or ssDNA or ssRNA, to induce duplex formations with “unprecedented” affinity (Vester & Wengel, 2004). In fact, affinity is so great, that care must be taken with regards to number of LNAs used, and placement relative to one another. Microarrays are well-suited for large-scale analyses, but has issues with discriminating single-nucleotide differences and varying probe characteristics (Wang & Yang, 2010). The advantage is that the melting temperature (Tm) between the capture probe and the short miRNA is increased as such to allow one equalized Tm for all miRNAs, for which the Tm values would range across 30ºC (Castoldi et al., 2007).
This normalized Tm also allows for higher discrimination between single-nucleotide polymorphisms (SNPs), and when used as an alternating monomer probe (Figure 5) has been shown to increase detection 30 to 50-fold (Vester & Wengel, 2004). The incorporation of LNAs into oligo probes is described as “mix-mers” (Castoldi et al., 2007) and have been incorporated into many miRNA detection methods, effectively both in vitro and in vivo (Válóczi et al., 2004; Castoldi et al., 2007). In a quantitative analysis of miRNAs in human tissue, and with a dual LNA probe variation, Neely et al. achieved results measuring as low as 500femtomolar concentrations (2006).
Another popular high-throughput detection method uses real-time RT-PCR with modified primers, illustrated in Figure 6 (Chen et al., 2005). The primers hybridize to the small miRNA; the base-stacking increases the Tm and the loop structure extends its physical size. Reverse-transcriptase creates cDNA, after which specific primers and a TaqMan dye-labelled probe are used with TaqMan PCR to quantify the miRNA (Chen et al., 2005). This method exhibits 1nt discrimination, minimizes DNA contamination, requires only 25pg of total RNA, can quantify up to 7 orders of magnitude, and is fast and accurate (Chen et al., 2005). Raymond et al. have had success with RT-PCR of the miRNA precursors (2005). Ro et al. developed a PCR method in which the miRNA is poly-adenylated, then a primer with oligo-dTs + adaptor is used to produce small RNA cDNA (srcDNA) as seen in Figure 7 (2006). Unfortunately, PCR technique is not feasible for small or rare samples, and multiplexing is not realistic.
After isolating and quantitating miRNAs using this modified PCR method, an additional cloning step can be added to increase potential of identification, as described by Ro et al. (2007). The 3’ and 5’ ends of the miRNAs are mapped via ribonuclease protection assay, after which the RNA probes are digested with RNase. Target mRNAs are predicted bioinformatically, and thus must be confirmed; therefore, a target validation vector is derived from a luciferase reporter vector. Pre-miRNA and putative target sequences are cloned into this vector, assayed with the oxidative bioluminescent luciferase, and luciferase activity is measured.
miRNA Identification
Finally, miRNAs must be identified; there are two major ways. Experimental identification, such as microarray or PCR, is efficient and reliable, with the potential to discover new transcripts or
specie-specific miRNAs; however, they are typically more expensive and require highly-skilled operators (Frazier & Zhang, 2011). The alternative computational strategy utilizes computer-based tools related to the prediction algorithms such as miRcheck and findMiRNA and capitalizes on an established basic local alignment search tool (BLAST) (Frazier & Zhang, 2011). Although fast an inexpensive, it is limited to whole-genome searches. Advantageously, neither methods require many new tools or software.
Once high-throughput results are obtained, expression profiles (in formats which are dependent on the detection techniques used) are created for comparison to one another, or with controls. These offer a condensed yet specific comparison, as the abundant number of miRNAs offer a highly unique profile (Figure 8). In one study, and supporting the potential strength of these analytical methods, it was found that 217 miRNA expression profiles were more accurate in classifying cancer tissue than were several thousand mRNA expression profiles combined (Lu et al, 2005).
Figure 8: An miRNA Heatmap Expression Profile after microarray (Liang et al., 2007)
Future Outlook
The future prospects for miRNA are bright indeed. miRNAs inherently offer two modes of action – target degradation or suppression – before any decision of application is even made. Adding more versatility is the option to use endogenous or non-endogenous miRNA for a specific target while minimizing secondary effects, or even avoiding them altogether. Furthermore, miRNAs are so small and elementary in nature that they can be synthesized while maintaining complete purity.
A major advantage of miRNA pertains to the field of genetic engineering (GE); the United States Department of Agriculture defines GE as “Manipulation of an organism’s genes by introducing, eliminating or rearranging specific genes using the methods of modern molecular biology, particularly those techniques referred to as recombinant DNA techniques,” and genetic modification must include “heritable movements” (2012). Using miRNA will avoid these designations.
In a world of increasing global movement, continually adapting bacteria and viruses, zoonotic disease, and increasing population, attention is rapidly being brought to efficient and effective means to combat disease and to confidently provide safe food. miRNAs offer alternative approaches to antibiotics in clinical and veterinary sciences, to using genetically-modified techniques in food production, and to older and less efficient means of genetic studies across all fields from which beneficial scientific data can be extrapolated. miRNA versatility will come from their simplicity.
Conclusion
miRNAs constitute new class of small, non-protein encoding transcripts with significant influence on post-transcriptionally regulated genetic expression. Since their discovery via classic genetic techniques, their roles are becoming clearer, miRNA biogenesis has become well understood across kingdoms, their modes of action are reasonably well identified, and a huge variety of analytical techniques and tools are emerging to increasingly improve their analyses. miRNAs have presented opportunities for careers in research and in industry, for novel projects, and most substantially, for scientific discovery.
Of course, with the recently discovered biological mechanisms and new tools, the full potential of exploiting and understanding miRNA is only preliminary. Results of studies in recent years have shown that prediction and detection methods are highly effective using expression profile comparisons. Such comparisons are vital in helping researchers understand the functions of miRNAs, but also for better understanding the system on which they have influence, to give a more complete functional genomic comprehension of this new class of RNA and the organism as a whole. From a basic genetic point of view, the journey of miRNA discovery can ideally provide a reminder for scientists to not overlook ostensibly insignificant topics.
Aside from the biological and evolutionary interpretation, miRNAs have immense implications in agricultural, clinical or industrial context; their high rate of conservation, high numbers of unique products and observed impact on genetic expression suggest a totally new area of genetic management. The specificity and epistatic nature is extremely attractive when compared with other intrusive and damaging alternatives used conventionally. And, although these transcripts are tiny, they will have a huge impact on scientific discovery.
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