Structural Biochemistry/Nucleic Acid/RNA/MicroRNA (miRNA)

Overview
MicroRNAs(miRNAs) are short, single-stranded RNAs that are about 21 nucleotides in length. Their function is to regulate gene expression. Like other types of RNA, miRNAs are transcribed from DNA; However, they do not participate in protein translation. miRNAs are non-coding RNAs that bind to complementary mRNA and inhibit their translation. miRNAs and siRNAs both function to interfere with gene expression. However, miRNAs are single-stranded, whereas siRNAs are double-stranded.

miRNAs have been determined to play a crucial role in regulation of DNA damage response. Scientists believe that the transmission of generic information in eukaryotic cells requires accuracy in DNA replication and chromosome as well as the ability to sense and repair spontaneous and induce DNA damage. In order to maintain genomic integrity, cells undergo a DNA damage response, a complex network of signaling pathways. This network is composed of coordinates sensors, tranducers and effectors in cell cycle arrest, apoptosis and DNA repair.

miRNAs have recently been linked to various diseases. Recent researches have shown that there is connection between dysregulation of miRNAs with certain diseases, which leads to the need of further researching in robust regulation of miRNA activity.

miRNAs once were considered to be very stable molecules because miRNAs expression is known to be strictly controlled by the mechanisms acting at the level of transcription and also the processing of miRNA precursors. However, recently, scientists have figured out another mechanism that is important for miRNA homeostasis which is the active degradation of mature miRNAs. Degradation of miRNA takes role in dynamic miRNA expression patterns. Researches showed that miRNAs degradation can have affect on specific sets of miRNAs even though how this specificity comes about still remains unknown.

Formation & Function
The main function of miRNAs is to regulate the translation of mRNA. In the nucleus, the miRNAs are first transcribed as primary miRNAs(pri-miRNAs) with caps and a poly-A tail. The pri-miRNAs are then processed into precursor miRNAs(pre-miRNAs) by an enzyme called Drosha. The structure of pre-miRNA is a 70 nucleotide-long stem-loop structure. The pre-miRNAs are then exported into the cytoplasm and split into mature miRNAs by an enzyme called Dicer. These mature miRNAs will integrate into the RNA-induced silencing complex(RISC) and activate the RISC. The activated RISC can then allow miRNAs to bind with the targeted mRNA and silence the gene expression. In animal cells, miRNAs are more commonly base paired with the mRNA and inhibit protein translation. The binding of miRNAs to complementary mRNA can degrade the mRNA and therefore terminate protein translation. Or miRNA can inhibit the reading of the 5'-cap and prevent translation. In plant cells, the miRNAs are more likely to perfectly bind with the target mRNA and promote cleavage. MicroRNA's are formed from the hairpins of long single-srranded RNA's that fold in on themselves. The double-stranded hairpins get cut by enzymes called dicer and results in a single-stranded microRNA. MircroRNA then forms a microRNA-protein complex and can then degrade a targeted mRNA and also block translation of targeted mRNA. In few instances, miRNA have shown signs of promoting translation, especially under starvation conditions. The reason for such activity are not known.

Canonical miRNA Function
Developing studies have demonstrated that miRNAs carry on a critical role in interacting with the canonical DNA damage response. The DNA damage response is an active system that includes commencement of transcriptional programs, enhancement of DNA restoration, and apoptosis if damage is severe. Breakages in DNA double-strands are mended by homologous re-fusion and non-homologous end-connecting repair pathways. Other forms of DNA damage are repaired by base excision repair (BER), nucleotide excision repair (NER), and DNA mismatch repair (MMR).

miRNA play a significant part in gene regulation and other cellular functions. Many important genes in the DNA damage response are managed by their corresponding miRNAs. For one, miRNAs monitor DNA damage response by way of target genes. In the process of DNA repair, chromatin remodeling takes place to permit DNA repair proteins access to DNA that are damaged. With more miRNA targets such as ATM, H2AX, and RAD52, DNA responsive genes are under inhibition by miRNAs. It has been revealed that higher expression of a certain miRNA -such as miR-421- will reduce ATM delivery, and downregulate H2AX in particular cellular situations.

DNA damaging agents in various treatments have proven to initiate miRNAs. Occurrences of DNA damage have depicted a correlation with the activation of miRNAs, underlying the significance that miRNAs regulate DNA damage response based on the magnitude of the DNA damage.

Noncanonical miRNA Function
Recent study has shown that by miRNA directly targeting the primary transcripts of other miRNA in the nucleus, it can control the biogenesis of the miRNA. A particular example is the miR-709 which negatively regulates the miR-15a/16-1. This particular miR709 is found in the mouse nucleus, and it binds specifically to miR15a/16-1 which are both 19-nucleotide recognition element. It clusters and blocks the processing of primary transcript of miR-15a/16-1 into the precursor. As such it regulates the maturation at a post-transcriptional level, which means post primary transcript but pre precursor. As such, because miR-709 can regulate the miR-15a/16-1, which in turn regulates the cell apoptosis, the miR-709 indirectly regulates the cell apoptosis. This in turn demonstrates that miRNA can affect the expressions of things within a cell because it can regulate the biogenesis of the other miRNA within the cells.

The miRNA can also regulate the long ncRNA. ncRNA are generally longer than 200 nucleotides and are non-protein-coding transcripts. The first experimental evidence that shows long ncRNAs are functional miRNA targets is shown by Hansen. In the experiment, the antisense transcript of the cerebellar degeneration related protein 1 (CDR1), which is a circular ncRNA, has been shown to be near perfect complementary with miR-671, which is in the nucleus. Within the experiment, miR-671 directs the cleavage of the CDR1 antisense transcript in an AGO2-dependent manner. Thus, with the negative regulation of the circular antisense ncRNA, it also decreases the CDR1 sense transcript. The study down shows that the antisense RNA can be destabilized by the miRNA through the AGO2 -mediated cleavage, and the sense mRNA can be stabilized by the circular noncoding antisense RNA.

miRISCs and Its Components
miRNA combine with Argonaute proteins and GW182 proteins to form miRNA-induced silencing complexes, or miRISCs. AGO attach to the N-terminus of the GW182 protein, while the miRNA bond to the AGO. The GW182 protein seems to be the more important of the two, as it contains the main silencing region. This was discovered when miRNA induced repression was still effective even after the knockoff of AGO in Drosophila cells.

miRNA Inhibition of Translation
miRNA possess several methods of inhibiting translation. Suppression can occur both before and post translation, although the former method seems to be preferred.

Before Initiation

 * 1) miRISC can suppress translation by interfering with the reading of 5' eIF4F-cap structure. The miRNA prevents the ribosome from reading the cap, thus initiation never starts. On the other hand, mRNA that were able to translate without the cap recognition step were not suppressed by the miRNA.
 * 2) miRNA are also able to prevent the creation of a functional ribosomal unit. On normal mRNA, the 40S and 60S ribosomal subunit come together to form the 80S complex, which helps translate the mRNA. miRNA inhibit 60S from joining with the 40S unit, making mRNA translation impossible. Translation is never able to start.

Post Initiation
The mechanisms for the three post initiation inhibitors are known.
 * 1) miRNA blocks the elongation of the new RNA being translated.
 * 2) The ribosome is forced to drop-off from the mRNA. The 40S and 60S ribosomal units split up before translation is complete.
 * 3) The miRNA induce preteolysis of the newly transcribed polypeptide chain. The chain is broken up by enzymes.

miRNA stability
In contrast to the suggestion in the past that miRNAs are highly stable, recent researches have shown that individual miRNAs in certain environments are subject to accelerated decay, which alters miRNA levels so that affects its activity.

During miRNA biogenesis, miRNAs are transcribed by polymerase II as primary transcripts (pri-miRNAs) and the are matured in a multi-step biogenesis process to produce the mature and functional miRNA form. In one case, the pri-miRNAs are captured by polyaldenylated and are quite long (several kilibases long). Pri-miRNAs possess hairpin structures which includes the mature sequence of miRNA in their stem. In another case, the precursor miRNAs (pre-miRNAs) can be kept in introns of mRNAs or other non-coding RNAs. In either of these two cases, the nuclear RNAse type III enzyme Drosha in a complex with co-factor DiGeorge syndrome critical region 8 homolog (DGCR8), cleaves near the base stem which releases about 70 nucleotides pre-miRNA.

Deadenylation and Decay
In deadenylation, the miRNA binds to AGO, GW182, and also poly(A)-binding protein (PABP). The PABP attaches to the GW182 protein, forming a slightly different miRISC. The miRISC removes the 5'-cap from the mRNA, which immediately causes decay of the mRNA. Deadenylation is effective because it rids the cell of excess mRNA, eliminating the chance of accidental translation. The decayed fragments are collected by the P bodies, and reused by the miRNA.

The degradation of miRNAs occurs under the aid of several miRNA-degrading enzymes. Many miRNA-degrading enzymes have been determined including both 3'to 5' and 5' to 3' exoribonucleases. Recent researches have shown that certain RNases were found to take the role in the turnover process of different sets of miRNAs in different organisms. However, the substrate specificity and phylogenetic conservation of individual miRNA turnover enzyme are still in the need of researching.

microRNA-206 and Synapse Repair
In a mouse model of ALS: When mice get ALS, production of microRNA-206 is induced/increased. Deficiency of microRNA-206 accelerates the progression of the disease. -MicroRNA-206 is required for regeneration of damaged neuromuscular synapses (the signals between muscle and nerve cells). When synapse is damaged, microRNA-206 turns on repair. Without miRNA-206, synapses cannot be repaired; however, some synapses can grow back. -MicroRNA-206 does this through histone deacetylase and fibroblast growth factor (FGF) signaling pathways. Growth factors are specific signals from other cells that tell the cell to grow. -MicroRNA-206 blocks translation. It then activates histone deacetylation which condenses chromatin, therefore blocks transcription. -MicroRNA-206 slows the progression of ALS by repairing neuromuscular synapses.

MicroRNA genes are found in intergenic regions. These regions have its own miRNA gene promoter and regulatory units. Approximately forty percent of miRNA genes are lie in the introns of the proteins coding, non-proteins coding, and even in the exons. The miRNAs are found in the orientation that are regulated together with its own host gene. Between forty-two to fifty percent of other miRNA genes were shown in a common promoter, which originate from polycistronic units. The polycistronic units have a discrete loops of 3-6 where the mature miRNAs are being processed, but the miRNAs family are not homologous structure function. Hence, the promoters have a few identical motifs to other genes promoters that were transcribed protein coding genes from the RNA polymerase II. Also, in the DNA template does not have the finish during the mature miRNA production, because there is about five percent of human miRNAs shows RNA editing. The site-specific modification of RNA sequences to yield products different from those encoded by their DNA. The yield of the product allows to increases the diversity, the scope of miRNA action implied from the genome alone.

Cancer
Recent studies have shown that miRNAs are involved in causing diseases. In the case of cancer, researchers found that miRNAs can inhibit the E2F1 protein that regulates cell proliferation. miRNAs bind to the mRNA first before translating the E2F1 protein. One microRNA, miR-21, was labeled as the first oncomir. It is known to aid in tumor growth and metastasis by targeting natural occurring tumor suppressors in the human body. Tropomyosin 1 (TPM1) is a direct target of miR-21, along with programmed cell death 4(PDCD4) and maspin, all of which are inversely correlated with the expression of miR-21 in the presence of tumors. This shows that miR-21 has the ability to target multiple genes and inhibit multiple metabolic pathways at the same time.

Kidney Fibrosis
Renal fibrosis is the excessive accumulation of fibrous tissues (connective tissues), occurring as a reparative process after scarring or trauma to the kidney. This type of nephropathy directly promotes renal dysfunction, which ultimately leads to kidney failure and death. Study has shown that a certain microRNA, miR-21, shows significant elevation in expression during the progression of kidney scarring. Experiments were conducted to validate this specific sequence and its effect in mice.

The abrogation of miR-21 in mice showed no overt abnormalities and no obvious suppression/prevention of tumor growths; however, these mice developed far less interstitial scarring tissue in response to kidney injury. Analysis has detected groups of genes and their subsequent metabolic pathways that were inhibited by miR-21. One of which involves peroxisome proliferator-activated receptor- α(Pparα), which is a lipid metabolism pathway that incorporates the synthesized anti–miR-21 oligonucleotides to inhibit miR-21. Pparα is found to ease the effects of ureteral-obstruction induced kidney fibrosis. miR-21 also regulates the redox metabolic pathway that involves a protein called Mpv171. The repression of Mpv171 in cells enhances kidney damage by reducing the production of oxygen radicals.

These studies demonstrate that miR-21 has a broad spectrum of influences on the microscopic scale and can be a suitable target for antifibrotic and cancer therapies.

Heart Disease
Another studies have shown miRNA inhibits the maturation in the murine heart, and plays an essential role during its development. The expression level of the miRNA is been changed in the disease of the human heart; it is the involvement in cardiomyopathies. During the heart disease development, they were several specific miRNAs that were been identified in animal models that were mostly in mice under pathological conditions. Those specific miRNA conditions key factors are important for cardiogenesis, the hypertrophic growth response, and cardiac conductance.