In the beginning when the first Micro RNA lin – 4 was discovered, it was thought that it was the only Micro RNA. This Micro RNA was acknowledged by genetics in the nematode Caenorhabditis elegans.
This Micro RNA is related to the development of the creature with time. Quite interesting to know about this gene is that, it is not responsible for encoding a protein itself, but generates two dissimilar codes for two tiny RNA molecules. These two RNA molecules are approximately 61 and 21 nucleotides in length.The first RNA molecule is the ancestor of the second RNA molecule which is of 21 nucleotides in length.
The larger one is capable of structuring another stem loop molecule which is stored in the nematodes.The smaller of the two RNA molecules controls the first Micro RNA lin – 4 by the formation of base paring. This is done to imperfect the opposite sites in the 30 un-translated areas or the UTR of the first Micro RNA lin – 4. In the start, the scientists regarded this phenomenon of gene silencing by a small RNA as a rare sight.
This was especially regarded to be rare in the case of nematodes.However, after the scientists discovered the second first Micro RNA, let-7, this view changed. This Micro RNA Let-7 is another type of small RNA that works for the developmental timing in small RNA that is also involved in developmental timing in Caenorhabditis elegans and also controls the first Micro RNA lin – 4.Both the first and the second Micro RNA’s, lin – 4 and let – 7 are known as small temporal RNA’s and are basically known for their part in developmental timing.
Out of these two RNA’s, let – 7 is more conserved in many multi cellular organisms than lin – 4. Even more, the second Micro RNA, let-7 is differentially articulated during development of bilateral animals.These findings clearly showed that the control and regulation of gene expression by these small RNAs was not limited to nematodes and was certainly a more general phenomenon that was expressed by various other multi cellular organisms. Until now, more than a hundred Micro RNA’s have already been discovered by the scientists in Caenorhabditis elegans and man.
The newly found Micro RNA’s are not listed according to the previous way – that is in a temporal specific method, like lin -4 and let – 7 were classified, but the new ones are expressed in tissue arrangement. These Micro RNA’s have been discovered in many other multi cell organisms and it is expected that the vertebrate organisms might have up to one thousand dissimilar Micro RNA’s.Further, it is also expected that these Micro RNA’s found in vertebrate organisms control almost 30% of the genes. This paper studies about the function of Micro RNA’s in animal development.
Micro RNA AND ITS MECHANISMThe Micro RNA biological genesis is divided amongst many various steps. At first the micro RNA genes are directly copied as RNA polymerase II which are in the form of a longer structured micro RNA. These RNA polymerase II are also called as Pri – Micro RNA’s. The development of the primary Micro RNA’s undergoes a step to step phase of growth and they are compartmentalized.
The Pri – Micro RNA’s are developed into a 80-75 -nucleotide in length Micro RNA’s with the help of an enzyme known as RNase III enzyme Drosha. The role of this RNase III enzyme Drosha, is to form a protein complex which has a double stranded RNA. This protein is called as DGCR8 (Pasha in flies). The Pri – Micro RNA’s are shifted from the nucleus by Exportin-5 in the existence of Ran-GTP as cofactor.
Within the cytoplasm, these Pri – Micro RNA’s are developed into 22 nucleotide in length, duplex Micro RNA’s with the help of another enzyme – RNase III enzyme Dicer. This enzyme – RNase III enzyme Dicer was first known for its function in RNA interference. In this interference, the RNase III enzyme Dicer, changes the doubly stranded RNA into small interfering RNA’a that also play a role in RNA interference.RNase III enzyme Dicer works together with the doubly stranded RNA to bind up the protein TRBP in Caenorhabditis elegans and Loquacious in Drosophila.
This process effectively brings closer the initiator and effectors steps of Micro RNA action. Next, the Micro RNA duplex are loosened and unwinded, and this process in initiated at the lowest possible lowest thermodynamic stability of the duplex end. The Micro RNA that is developed at this stage is the future Micro RNA and is also known as the GUIDE RNA. These mature Micro RNA’s areincluded into a ribo-nucleoprotein complex, which is known as miRNP, and is comparable to the to the RNA-induced silencing complex or RISC, which is the effector of the RNA interference.
However, ribo-nucleoprotein complex, miRNP and the RNA-induced silencing complex are not identical in nature. In RNA-induced silencing complex, the Micro RNA’s can intervene in the lower regulation of the target gene activity in two different methods or modes:Translational inhibitionTarget mRNA cleavage.However, the selection of the method / mode is made according to the level of complementarities, amongst the Micro RNA and the target gene in amalgamation with an Argonaut family protein. If there are high results of complementarities between Micro RNA and the target gene, then the second mode is selected – Target mRNA cleavage.
This is followed by common RNA degradation of the gene target. If in case the results of complementarities between Micro RNA and the target gene are low, or there is partial complementarity, then it results in the selection of translational inhibition. The scientists are still not aware of the function of the translational inhibition mode. It is only suggested at the time being that the Micro RNA’s target the genes and these genes could be seized on polysomes.
Another suggested function of the translational inhibition mode is that the genes be employed by the P-bodies where they are used up of the translation machinery and are in the process worn out and degraded. In disparity to the plant kingdom, Micro RNA’s brought about the Target mRNA cleavage in animals, for example the Target mRNA cleavage of HOXB8 mRNA by miR-196 in mouse embryos. However, this is much less common in Translational inhibition. Argonaut proteins are significantly in attendance in the RNA-induced silencing complex or RISC and all the Argonaut proteins that are present in mammals, actually do the binding up between the Micro RNAs.
On the other hand, in both mammals and Drosophila, Argonaut 2 proteins is the only main catalytic locomotive of RNA-induced silencing complex or RISC that act as a go-between the targeted RNA cleavage. Furthermore, Drosophila Argonaute1 and the two Argonaute1 homologs alg-1 and alg-2 in C. elegans are required for miRNA production, stability and function. BIOLOGICAL FUNCTIONS OF Micro RNA’s IN ANIMALSThe Micro RNA’s perform multiple biological processes.
Several experiments have shown the various functions that are performed by the Micro RNA’s. Developmental timing in worms – the first two Micro RNA’s lin – 4 and let – 7, were studied and it was found that they have a role to play in the hetero-chronic pathway, which controls the developmental timing in C. elegans. Let – 7 was found to be a suppressor of lin-14 mutants.
This clearly showed that the Let -7 acts in some way. If there occurs a loss of function mutation in let 7 and lin 4, then both of them experience retardation but at different developmental stages. The null mutants of Lin 4, repeat detailed upshots of the primary larval phase at following later on stages, let-7 null mutants repeat larval cell upshots at the mature phase. On the other hand, over expression of let-7 provides intelligent growth, the contradictory heteroc-hronic phenotype.
The timing of the phenotypes keep up a correspondence with the onset of lin-4 and let-7 expression, which is early and late in improvement, in that order. Thus, lin-4 is an early developmental timer and let-7 a late developmental timer.Neuronal asymmetry in worms – “A surge of genes, concerning two micro RNAs, settle on the issue of Neuronal left/right asymmetric expression of chemosensory receptorgenes in the left (ASEL) and right (ASER) chemosensory neurons of C. elegans.
The lsy-6 miRNA is expressed in the ASEL neuron and inhibits the expression of its target, the Nkx-type homeobox gene cog-1. This ultimately leads to the expression of the GCY-7 chemosensory receptor in ASEL. In the ASER neuron, miR-273 inhibits the translation of die-1 mRNA. DIE-1 is a zinc-finger transcription factor needed for the transcription of lsy-“.
Therefore, the expression of miR-273 leads to the down regulation of lsy-6 and subsequently to the expression of the GCY-5 chemosensory receptor in ASER . Thus, inverse and sequential expression of two micro RNAs leads to asymmetric expression of chemosensory receptors in neurons of C. elegans. (Lau, N.
C., Lim, L.P., Weinstein, E.
G. and Bartel, D.P. (2001), abundant class of tiny RNAs with probable regulatory roles, 294, 858–862.
) Vertebrate development – more than a few observations demonstrate that micro RNAs are necessary for the standard growth of mammals. initially, mouse and human cells state a particular set of micro RNAs that are down regulated over differentiation into embryo bodies. Secondly, the cells that are lacking in dicer are workable, other than do not shape up in mature micro RNAs and they do not succeed to set apart in vitro and in vivo.First, most of the Micro RNA’s were discovered by the loss / gain processes of the genetic screens in Caenorhabditis elegans and Drosophila.
Given that, most of these micro RNA’s have been identified by their functions, it is likely to be accurate that they are involved in tissue differentiation. The other functions that have been identified of the Micro RNA’s seem to originate form the reverse genetic approaches. These approaches include likes of Micro RNA’s knockout or knockdown and micro RNA’s over expression studies. In addition, the micro RNA’s appearance outline, are mainly determined by, the various experiments like for example, the microarray- and in situ analysis experiments have shown that there are some special Micro RNA’s with specific expression designs which give hints to the functions of some special micro RNA’s.
According to the scientists there are only seven micro RNA target genes have been discovered by reverse and forward genetic screens. Many of the target genes are discovered by computational predictions. Recent study and research shows that for every Drosophila Micro RNA, there are an average of 100 various / dissimilar target genes. Most of these target genes are regulated within the seed site interactions.
GENERAL FUNCTION OF Micro RNA’s IN ANIMALS Until so far, these micro RNA’s have only been discovered in multi-cellular organisms and are not found to exist in single cell organisms. This fact confirms that the presence of Micro RNA’s is required for tissue differentiation. Many tests and experiments have proved that this Micro RNA’s are indeed the regulators behind the cell / tissue differentiation. Firstly, since poorly formed or poorly differentiated cells do not need Micro RNA’s in order to exist and this is quite obvious from the detailed information that mouse cells that do not have Micro RNA’s are feasible but be unsuccessful to differentiate and the zebra fish germ cells do not need micro RNA’s for their survival and contribution to the germ line.
Secondly, a number of Micro RNA’s are not present in the early samples of zebra fish, when the cells of the zebra fish were not differentiated. Although the cells of Zebra fish have tissue specific categories developed at a later stage. Third, since the absence of the Micro RNA’s are also not necessary for tissue fate foundation, for the duration of early zebra fish growth, but are necessary for later on improvement steps and tissue development / functioning. Fourth, numerous kinds of human cancer cells have condensed the Micro RNA’s appearance as compared to their complete distinguished tissue at the time of origin.
Fifth, the animals possessing the vertebrae, program hundreds dissimilar micro RNAs that are anticipated to control up to 30% of the genes. Sixth, the micro RNAs posses a larger number of molecular abundance in each cell. In Caenorhabditis elegans there are some micro RNA’s that are predicted to ne present in as much as 55000 copies in each cell. This estimate is at least 500 times higher than the level estimated in a characteristic worm.
The autonomous formation of micro RNAs is identified by the elevated level of assortment that is linking the numerous micro RNA families of the plant and animal micro RNAs. In addition, a small number of DNA base-pair alterations that have been discovered, corresponding to the seed sequences of micro RNAs and are expected to change the collection of target genes. In corresponding, the target genes can without difficulty be included underneath the power of micro RNAs in the course of changes of only a few base-pairs in the DNA. Single micro RNAs possess the ability to regulate over a hundred target genes and the mixed action of micro RNAs is anticipated to control the face of thousands of micro RNAs.
The controlling of such a high number of genes is actually important for the purpose of tissue differentiation. Moreover, the post-transcriptional ruling of the gene expression can also control the variations that are recorded in gene expression. These results of controlling the target genes leads to the creation of stable protein levels, which might also be required to keep cells differentiated. CONCLUSIONSEver since the first of the Micro RNA lin – 4 and let – 7 were discovered by the scientist it has also been proved that these Micro RNA’s form a significant part and a major category of post transcriptional gene controllers.
These micro RNA’s are present widely in all multi cellular organisms, from plants, animals to humans. A majority of animals’ posses about hundred of Micro RNA’s of which most of them are unknown or their functions are unknown to the scientists. However, the limited number of Micro RNA’s that are known to the fraternity, already show signs that these Micro RNA’s possess ultimate abilities to perform in various biological processes. Moreover, in animals like mouse and zebra fish, which do not possess the Micro RNA’s or their production, reveal that Micro RNA’s are required for the development of vertebrae and are responsible for the division and control of tissue and cells.
The current existing sets of Micro RNA’s that are known to the medical science are known to regulate the millions of target Micro RNA’s up to 30%. This figure may still move up because a large number of Micro RNA’s and their functions are still unknown to the scientists. The confirmation of the continuation of these micro RNA’s and their interface with target genes will be the solution to discover the role of all individual micro RNAs during expansion, disease and other cellular processes. REFERENCES Lee, R.
C., Feinbaum, R.L. and Ambros, V.
(1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854.  Wightman, B.
, Burglin, T.R., Gatto, J., Arasu, P.
and Ruvkun, G. (1991) Negative regulatory sequences in the lin-14 30- untranslated region are necessary to generate a temporal switchduring Caenorhabditis elegans development. Genes Dev. 5, 1813– 1824.
 Reinhart, B.J., Slack, F.J.
, Basson, M., Pasquinelli, A.E., Bettinger, J.
C., Rougvie, A.E., Horvitz, H.
R. and Ruvkun, G. (2000) The 21-nucleotide let-7 RNA regulates developmentaltiming in Caenorhabditis elegans. Nature 403, 901–906.
 Slack, F.J., Basson, M., Liu, Z.
, Ambros, V., Horvitz, H.R. and Ruvkun, G.
(2000) The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA andthe LIN-29 transcription factor. Mol. Cell 5, 659–669.
 Pasquinelli, A.E., Reinhart, B.J.
, Slack, F., Martindale, M.Q., Kuroda, M.
I., Maller, B., Hayward, D.C.
, Ball, E.E., Degnan, B., Muller, P.
, Spring, J., Srinivasan, A., Fishman, M., Finnerty, J.
, Corbo, J., Levine, M., Leahy, P., Davidson, E.
and Ruvkun, G. (2000) Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 408, 86–89.  Lau, N.
C., Lim, L.P., Weinstein, E.
G. and Bartel, D.P. (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans.
Science 294, 858–862.  Lagos-Quintana, M., Rauhut, R., Lendeckel, W.
and Tuschl, T. (2001) Identification of novel genes coding for small expressed RNAs. Science 294, 853–858. Wienholds, E.
, and Plasterk, R. (2005). MicroRNA function in animal development. FEBS.
(579): p911-5922. Plasterk, R. H.A.
(2006). Micro RNAs in animal development. Cell.Vol.
124: p877-881. Abbot Allison, et al. (2005). The let-7 MicroRNA Family Members mir-48, mir-84, and mir-241 Function Together to Regulate Developmental Timing in Caenorhabditis elegans.
Developmental Cell. Vol. 9: p 403-414.