Microrna Biogenesis: Epigenetic Modifications as Another Layer of Complexity in the Microrna Expression Regulation

Since their discovery, microRNAs have led to a huge shift in our understanding of the regulation of key biological processes. The discovery of epigenetic modifications that affect microRNA expression has added another layer of complexity to the already tightly controlled regulatory machinery. Modifications like uridylation, adenyla-tion and RNA editing have been shown to have variable effects on miRNA biogenesis and action. Methylation of the N6 adenosine has been studied extensively in mRNA. Presence of the N6-methyl-adenosine (m 6 A) mark and its critical importance in miRNA biogenesis in animals adds to our understanding of the regulatory mechanisms, while its effect on miRNA biogenesis in plants is yet to be understood.


INTRODUCTION
The discovery of small non-coding RNAs and their subsequent characterisation as keysequence specific regulators of a variety of eukaryotic processes has led to the addition of another dimension in our understanding of gene regulation.Micro-RNAs (miRNAs) are a major part of the group of such small non-coding RNAs which includes a variety of other regulatory RNAs, like small interfering RNAs (siRNA), PIWI interacting RNAs (piR-NA) etc. MiRNAs are ~21nt long RNA molecules that specifically repress their target mRNAs.They were first discovered in Caenorhabditis elegans and were identified as key regulators of genes that are responsible for various developmental events (Lee et al., 1993;Wightman et al., 1993;Reinhart et al., 2000).Although since then over-expression or under-expression of miRNAs has been associated with a number of diseases (Li et al., 2012), the mechanisms regulating miRNA expression, including their biogenesis and mode of action, are still not fully understood.

MicroRNA BIOGENESIS
Although the major function of miRNAs in animals and plants is similar, the biological pathway of their biogenesis varies considerably.The differences are visible beginning with the structure and location of the miRNA genes (MIR genes).Animal miRNAs are mainly located within the introns of protein coding or non-coding RNA genes (80%) (Kim et al., 2009).As opposed to animals, in plants most of the miRNA genes are exonic and are transcribed by independent transcription units (roughly 90%) (Szarzynska et al., 2009;Kruszka et al., 2013;Szweykowska-Kulinska et al., 2013;Alaba et al., 2015).These genes vary vastly in length and may contain introns.Studies in humans and Arabidopsis thaliana show that splicing and complex alternative splicing events play a significant role in proper regulation of miRNA biogenesis (Laubinger et al., 2008;Bielewicz et al., 2013;Schwab et al., 2013;Agranat-Tamir et al. 2014;Mattioli et al., 2014).In both, animals and plants, the MIR genes have been shown to be clustered together, although the animal miRNA genes display considerably higher clustering (Merchan et al., 2009;Axtell et al., 2011).

Animal miRNA biogenesis
In animals, most of the miRNAs are products of the RNA polymerase II transcription, but some human miRNAs have been shown to be products of the RNA polymerase III activity (Borchert et al., 2006).Animal pri-miRNAs, bearing miRNA/miRNA* in the hairpin structure, are further processed in a two-stepreaction that is completed in the nucleus and the cytoplasm, and involves two different types of the RNase III enzymes.The initial cleavage, resulting in the formation of pre-miRNA molecules, is completed in the nucleus by DROSHA RNase III accompanied by DGCR8 and other proteins that together form a Microprocessor complex.Pre-miRNAs that already contain the mature ends of miRNA/miRNA* duplex at the opposite end of the hairpin loop structure, are then exported to the cytoplasm by Exportin 5.In the cytoplasm, another RNase III enzyme called DICER cleaves the pre-miRNA to release the miRNA/miRNA* duplex (Lee et al., 2003;Yi et al., 2003;Denli et al., 2004).

Plant miRNA biogenesis
In plants, RNA polymerase II is involved in production of the primary miRNA (pri-miRNA).These pri-miRNAs vary in length and contain a hair-pin loop structure, housing the miRNA/miRNA* duplex (Xie et al., 2005).Pri-miRNAmolecules are substrates for an RNase III type enzyme, DICER Like 1 (DCL1).DCL1, accompanied by a HYPONASTIC LEAVES 1 (HYL1), SERRATE (SE) and other proteins form the plant Microprocessor, which cleaves the pri-miRNA molecules in two steps.The first step leads to the formation of an in-termediary pre-miRNA, and finally the miRNA/miRNA* duplex is released.In contrast to the animal miRNA biogenesis, the initial DCL1-promoted pri-miRNA cleavage does not generate the mature ends of miRNA/miRNA* duplex at the opposite end of the pre-miRNA hairpin loop structure.Unlike in animals, all of these events take place in the nucleus in specialised sub-nuclear regions called the D-bodies, and the DCL1 RNase is the only RNase involved in generation of the miRNA/miRNA* duplexes.The miRNA/miRNA* duplex is then exported from the nucleus by the plant homolog of Exportin-5 called HASTY (Vazquez et al., 2004;Park et al., 2005;Kurihara et al., 2006;Fang & Spector, 2007).
These miRNA/miRNA*duplexes, both in plants and animals, are then loaded onto the AGO proteins to form an RNA Induced Silencing Complex (RISC) where they selectively regulate the protein expression by either translational inhibition or mRNA degradation (Vaucheret et al., 2004;Baumberger & Baulcombe, 2005;Qi et al., 2005;Treiber et al., 2012).
Many other proteins that are required for the biogenesis of miRNAs have been identified.These proteins are rather specific for animals or plants (for review see: Chen, 2005;Kim, 2005;Winter et al., 2009;Voinnet, 2009;Krol et al., 2010;Ha & Kim, 2014).

miRNAs TARGET GENE INTERACTION AND REGULATION
Two major mechanisms of gene regulation via miR-NAs are known, and these mechanisms depend on the degree of complementarity between the miRNA and the target mRNA.A high degree of complementarity between the miRNA and its target mRNA results in mRNA cleavage by the RISC complex.A lower degree of self-complementarity leads to regulation via translational inhibition (Zeng, 2003;Meltzer, 2005).As miR-NAs can regulate targets with low self-complementarity, one miRNA can regulate more than one target and vice versa (Meltzer, 2005).

In animals
Animal miRNA binding sites in 3'UTRs are the most common, although miRNA binding sites have been shown to be present in 5'UTRs and the coding regions as well (Lewis et al, 2003;Lytle et al, 2007;Tay et al., 2008;Carthew & Sontheimer, 2009;Schnall-Levin et al, 2010).Often, animal miRNA molecules are not highly complementary to their target mRNA and the prevalent gene regulation mechanism is translational inhibition.Translational inhibition can occur at the translation initiation stage (by inhibition of ribosomal assembly) or post initiation stage (by early dissociation of the ribosome assembly) (Eulalio et al, 2008;Filipowicz et al, 2008;Chekulaeva & Filipowicz, 2009;Huntzinger & Izaurralde, 2011;Hussain, 2012).Although the translation inhibition mechanism is vastly prevalent in animals, it cannot be assumed to be the only one (Yekta et al, 2004;Millar & Waterhouse, 2005).Imperfect complementarity between the miRNA and its target does not lead to direct cleavage of the target, but it can cause the target degradation indirectly via deadenylation.The RISC complex along with the poly-A binding protein are important for deadynalation of mRNA, which is followed by mRNA decapping and degradation (Bagga et al, 2005;Eulalio et al, 2009;Hussain, 2012).In another example, mammalian miRNA196 has been shown to have perfect complementarity with its target and to directly cause the cleavage of its target mRNA (Yekta et al, 2004).

In plants
Plant miRNA target sites are found within ORF's, 5'UTRs, 3'UTRs as well as noncoding transcripts (Sunkar & Zhu, 2004;German et al, 2008;Axtell et al, 2011).Plant miRNAs are almost perfectly complementary to their target mRNAs.The miRNA induced target mRNA cleavage has been shown in many examples.However, this process is thought to overlap with translational inhibition, as was shown in the case of APATELA2 (AP2).AP2 has one to zero mismatches with the miRNA172, and yet appears to be regulated by translational inhibition, although some slicing also takes place (Aukerman & Sakai, 2003).This points towards a more complex mechanism of gene regulation by miRNAs where interplay of both mechanisms exists.

POST-TRANSCRIPTIONAL MODIFICATIONS DURING miRNA BIOGENESIS
After transcription, the miRNA processing-intermediates, as well as mature miRNAs, undergo a variety of modifications.These modifications may or may not have a direct effect on the miRNA action.Some of these modifications include uridylation, adenylation, RNA editing and methylation.

Uridylation and adenylation
In animals, most of the miRNAs are not methylated.These unmethylated miRNAs are usually uridylated at the 3'end by Terminal Uridylyl Transferases (TUTase), as was shown in the case of let-7, miR-10, miR-99/100, miR-196 and miR125 families of miRNAs (Yu et al., 2005;Hagan et al., 2009;Thornton, 2014).Some miR-NAs can also be tailed by adenylation.In mice, miRNA 122 has been shown to be adenylated by Germ Line Dependent 2 (GLD-2) after unwinding of the miRNA duplex (Katoh et al., 2009).Uridylation has been shown to be a negative regulator of the miRNAs' activity, where mono-uridylation inhibits further processing and polyuridylation promotes degradation; adenylation has been shown to have both, positive and negative regulatory effects (Heo et al., 2008;Katoh et al., 2009;Backes et al., 2012).
In Arabidopsis, HEN1 SUPPRESSOR1 (HESO1) has been shown to uridylate the unmethylated miRNAs.The null mutants of HESO1 have been shown to carry increased amounts of miR166/165, miR169, miR171/170 and miR172, while miR173 level was increased in the HEN1 null mutant background (Ren et al., 2012).Non template adenylation has been also detected in Populus trichocarpa miRNAs where it was shown to increase the miRNA stability.The factors responsible for adenylation are not fully understood yet (Lu et al, 2009).

RNA editing (A→I)
Another type of modification called RNA editing involves modification of adenosine residue into inosine (A→I).Adenosine Deaminase Acting on RNAs (ADAR) act on double stranded RNAs and convert adenosine into inosine.The most common target RNA molecules for such editing are those that contain repetitive elements, like Alu and Lines located within introns and 3'UTRs.Nearly 20% of human pri-miRNAs have been shown to be affected by A→I editing.Pri-miRNA 142 and 151 were shown to undergo this kind of editing, which blocked further processing of these pri-miRNAs and their consequent accumulation (Yang et al., 2006;Kawahara et al., 2007).The A→I editing has been also shown to change the target specificity of miRNA 376, where the edited miRNAs target a different set of mRNAs.In this case, the A→I conversion does not hamper further processing of the pri-miRNA but results in the production of mature miRNAs with an altered sequence, and thus leads to targeting of a completely different set of genes (Kawahara et al., 2007).

Methylation at the 3' end
In plants, HUA Enhancer I (HEN1) methylates the 3' nucleotides at the 2'O position.This happens before the miRNA/miRNA* complex is exported out of the nucleus and prevents the non-templated 3' uridylation (Li et al., 2005).Methylation by HEN1 protects miRNAs, as well as siRNAs, from the HESO-1 directed uridylation, a modification that leads to miRNA instability (Yu et al., 2005;Li et al., 2005).

N6 Adenosine methylation
A more ubiquitously present modification in almost all types of RNAs is the methylation of N6-adenosine.It is the most abundant in mRNAs, where it is found in almost 3-5 sites per mRNA molecule in mammals and plants.These modifications are abundant in the 3'UTR and near the stop codons (Schibler et al., 1977;Bokar et al., 1994;Meyer et al., 2012;Bodi et al., 2012;Pan, 2013).The m 6 A modification has been shown to be reversible, and numerous m 6 A containing mRNAs have been mapped by transcriptome wide mapping.Human MET-TL3 (or MTA70) and plant mRNA adenosine methylase (MTA), homologs of yeast Inducer of Meiosis 4 (IME4), have been shown to introduce the m 6 A modification at a specific sequence motif: RRACH, where R=G/A and H= A/C/U (Wei et al., 1976;Bokar et al., 1997;Clancy et al., 2002;Bodi et al., 2010;Jia et al., 2013).Knockout of either theMETTL3 gene in the animal cell lines or the MTA gene in plants has been shown to be lethal and this may indicate a crucial regulatory role of m 6 A modifications (Bokar et al., 2005;Zhong et al., 2008).The discovery of two human RNA demethylases, Fat Mass and Obesity Associated Protein (FTO) and Alpha-Ketoglutarate-Dependent Dioxygenase homolog 5(AlkBH5), may indicate that this modification may be under a spatial or temporal control (Jia et al., 2011;Zheng et al., 2013).FTO and ALKBH5 are homologues of the non-heme Fe(II)/α-ketoglutarate (α-KG)-dependent AlkB family of de-oxygenases, and are known to catalyze demethylation.These proteins are conserved in vertebrates and null mutants of FTO in mice have been shown to cause various developmental defects, including lower body weight, reduced fat mass and increased postnatal lethality.Knockout of ALKBH5 causes reduced fertility while also causing an increase in the m6A content in RNA.
MicroRNA transcripts have been also shown to carry the m 6 A modifications.RNA immunoprecipitation with m 6 A antibodies and the presence of the consensus RRACH sequence in pri-miRNAs provide a strong evidence of m 6 A presence in the human miRNAs (Berulava et al., 2015).It was also shown that knockdown of the FTO gene resulted in altered steady state levels of miR-NAs (Berulava et al., 2015).Recently, the m 6 A modification was shown to act as a mark for animal pri-miRNA processing.It was shown that DGCR8 identifies the m 6 A mark and then recruits DROSHA to cleave the pri-miR-NA and yield pre-miRNA.The m 6 A modification was shown to be enriched in the pri-miRNAregions proximal to the pre-miRNA sequences (Alarcón et al., 2015).A recent study revealed that a nuclear reader protein HNRNPA2B1 mediates recruitment of the microprocessor machinery for further processing.HNRNPA2B1 was shown to bind directly to the m 6 A mark, enhancing the binding of DGCR8 to pri-miRNA transcripts and affecting the miRNA biogenesis in a manner similar to MET-TL3 (Alarcón, C.R. et al., 2015).Figure 2 summarizes the action of METTL3 in animal miRNA biogenesis.
Almost nothing is known about the influence and abundance of m 6 A in the plant miRNA precursors.In Arabidopsis thaliana, the m 6 A mark is abundant in mRNA and has been shown to be enriched around the start codons, as well as the 3'UTR and stop codons.Plants with reduced level of METTL3 (MTA70) display highly altered phenotypes (Bodi et al., 2012;Luo et al., 2014).A homolog of human HNRNPA2B1 is also present in Arabidopsis, but nothing is known about its role in the miRNA biogenesis.

CONCLUSIONS
The presence of m6A in mRNA has been studied extensively and although the mechanism of its action is not well known, it is clear that it plays important roles in regulation of various metabolic functions.The discovery of two m6A mRNA demethylases in humans provides us with some evidence that this modification is reversible and thus brings up many more questions regarding the dynamics of this modification.The recent discovery of m6A as a mark for pri-miRNA processing adds miRNAs to the group of RNAs affected by this modification.It is imperative for us to know how big is the effect exerted by m6A modifications on the global RNA levels, and how its absence or presence changes the dynamics of gene regulation.
With significant discoveries being made in the context of m 6 A modifications in the animal miRNAs, the time is apt to know more about this modification and its implication in plants.We already know that the m 6 A mark in mRNAs is critical for the proper development of plants.To know if the effect of m 6 A modification extends to the miRNA level and how it regulates the plant miRNA biogenesis is an exciting new avenue in research.Does the m 6 A mark work in the same way as it does in humans and marks the pri-miRNA for further processing, and are there any mediators like HNRNA2B1 involved?These are some of the questions that are waiting to be answered.With the interest in the m 6 A mark in plant miRNA just gathering pace, we are sure to have some answers in the near future.The pri-miRNA is marked by METTL3 with the m 6 A mark.HNRN-PA2B1 reads the m 6 A mark and promotes binding of DGCR8, which helps in efficient recruitment of the Microprocessor.

Figure 1 .
Figure 1.MicroRNA biogenesis overview: (a) Animals: The MIR genes can be transcribed by either RNA polymerase II or III.DROSHA, together with the DGCR8 protein, cleaves the pri-miRNA to form pre-miRNA, which is exported by EXPORTIN5 to the cytoplasm, where DICER cleaves pre-miRNA to release the miRNA/miRNA* duplex.miRNA/miRNA* duplex is then incorporated into the AGO proteins, forming the RNA induced Silencing Complex.Gene expression is regulated by recognition of the target mRNA by RISC via sites present mainly in the 3'UTR of mRNA.Translation inhibition is the major mechanism, while some examples of degradation by slicing have been also observed.(b) Plants:The MIR genes are transcribed by RNA polymerase II.Pri-miRNA is processed by DCL1, leading to the formation of intermediary pre-miRNA and finally miRNA/miRNA* complex.HEN1 methylatesmiRNA/miRNA* duplex at the 3' ends, protecting it from HESO1 mediated uridylation.miRNA/miRNA* duplex is then exported to the cytoplasm by HASTY, where it is incorporated into AGO proteins to form the RNA induced Silencing Complex (RISC).Target sites are present within the ORFs, 5'UTRs and 3'UTRs.miRNAs are highly complementary to the target mRNAs, and gene expression is regulated by the mRNA degradation pathway, although translation inhibition has been also observed.

Figure 2 .
Figure 2. Role of METTL3 and HNRNPA2B1 in miRNA biogenesis in animals:The pri-miRNA is marked by METTL3 with the m 6 A mark.HNRN-PA2B1 reads the m 6 A mark and promotes binding of DGCR8, which helps in efficient recruitment of the Microprocessor.