MicroRNAome

From Omics.org

Discovered just over a decade ago, appeals of small noncoding ribonucleic acid (ncRNA), especially on micro ribonucleic acid (miRNA), become hottest topics in biological communities.  It still unsolved mysteries of molecular mechanism and involved with diverse areas of transcriptiome and molecular activities.  We believe this regulatory small ncRNA are recognized as one of the major regulatory gene families in eukaryotic cells.  Hundred of those small ncRNA genes have been found in animals, plant, and viruses.  In this chapter, it deals with heavy of biological background perspective for ncRNA, especially on miRNAs.  This summarizes the current status of known miRNA gene mechanism, function, and expression profiling.

A brief history of microRNA discovery


The history of the miRNA discovery is an example of chance, serendipity, and perseverance.  It is also a success story for open science communication and close collaboration among scientists.  A much smaller number of ncRNAs are initially identified genetically.  The gene line-4 and let-7 in Caenorhabditis Elegans are two such genes5.  line-4 was first isolated in a mutant screen and a characterized as having “abnormal post-embryonic cell lineages”6.  Using classical epitasis analysis, Ambros later showed the line-4 is upstream for the genes line-14 and line-287.  Later experiments showed that line-14 is downregulated through its 3’ untranslated region, UTR, and that line-4 is necessary for this regulation8.  From this work, Rubkun and colleagues suggested the simple model that the lilne-4 gene product directly interacts with the line-14 UTR8.  In working to clone the line-4 gene, Lee, Feinbaum, Ambros rigorously narrowed down the genomic interval of interest and systematically mutated all ORFs there to demonstrate that the line-4 gene product is not a protein.  Instead, they found the line-4 gene product is a small ncRNA that shows antisense complementarily of the line-14 3’-UTR9.  A later study found and cloned a gene, let-7, that appears to encode a similar small ncRNA that functions in a similar manner1.  Many more of these miRNAs have been found in studies that will be discussed on this paper as a major topic. 

Biogenesis of microRNAs


According to the current convention, a miRNA is defined as 19-25 nucleotides in length, which is generated by the RNase-III-type enzyme Dicer from an endogenous transcript that contains a local hairpin structure13.  This evidence brings to attend the transcription miRNA genes are mediated by the RNA polymerase II (poly II).  The primary transcripts of miRNAs (pri-miRNAs) contain a 5’ 7-methyl guanosine cap and a 3’ poly-A tail, modifications that are trademarks of the poly II transcription 2-4. The association of miRNA with poly II, the miRNAs are under elaborate control of various regulation factors in different development stages and tissues.  It is becoming evident that miRNA expression profiles are indeed complicated.  It should bear in mind, though, that there is still possibility that a small portion of miRNAs are transcribed by poly III.  And so far the DNA elements that are common to most poly II promoters such as that TATA box and the TFIIB recognition elements have not been identified for miRNAs.

Overviews of the model of miRNA biogenesis4 (shown in figure 1), miRNA genes are transcribed by RNA polymerase II (poly II) to generate the primary transcript (pri-miRNAs). The initiation step (‘cropping’) is mediated by the Drosha complex (also known as the Microprocessor complex).  Drosha is located mainly in the nucleus.  The product of this unclear processing step is a ~70-nucleotide (nt) pre-miRNA, which possesses a short stem plays a ~2-nucleotide 3’ overhang.  This structure might serve as signature motif that is recognized by the nuclear export factor exportin-5.  Pre-miRNA constitutes a transport complex together with exportin-5 and it cofactor Ran (the GTP-bound form).  Following export, the cytoplasmic RNase III Dicer participates in the stand is selected as the mature miRNA, whereas the other strand is degraded.  Pre-miRNAs are transported to the cytoplasm by exportin 5 and are processed into miRNA:miRNA duplexes by Dicer.  Dicer also processes long dsRNA molecules into small interfering RNA (siRNA) duplexes.  Only one strand of the miRNA:miRNA duplex or the siRNA duplex is preferentially assembled into the RNA-induced silencing complex (RISC), which subsequently acts on its target by translational repression or mRNA cleavage, depending, at least in part, on the level of complementarily between the small RNA and its target.


 

Genomic Location of microRNAs



A recent analysis of miRNA gene locations relative to known transcription units brought to light new issues regarding miRNA biogenesis10.  By combing up-to-date genome assemblies and transcription unit databases, Bradley and colleagues showed that ~70% of mammalian miRNA genes are located in defined transcription units11 (shown in figure 2).  Interestingly, 117 miRNA genes were found in the introns the sense orientation, which is more than previously expected.  Of these 117 intronic miRNAs 90 miRNAs are in the introns of protein – coding genes, whereas 27miRNAs are in the introns of ncRNAs.  A further 30 overlaps with the exons of ncRNAs and 14 miRNAs overlap with either an exon or an intron depending on the alternative splicing pattern.  Therefore, miRNA genes can be grouped on the basis of their genomic annotation (shown in figure 2) this following10-11:

         First, exonic miRNA in non-coding transcription units

         Second, intronic miRNA in non-coding transcription units

         Third, intronic miRNA in protein-coding transcription units

In addition, there are miRNAs that overlap with either an exon or an intron depending on the alternative pattern and are labeled as “mixed” category.  Furthermore, it is noticed that s small number of human miRNAs are found in 3’ and 5’ UTR of coding mRNA.  In mammalian, a subset of conventional miRNAs is derived from as well as genomic repeats12-13.

Function of microRNAs

miRNA function as guide molecules in post-transcriptional genes silencing by base pairing with target mRNAs, which lea to mRNA cleavage or translational repression.  With >200 members pre species in higher eukaryotes, miRNAs are one of the largest gene families, accounting for ~1% of the genome11, recent studies have revealed that miRNAs have key roles in diverse regulatory pathways, including control of developmental timing, Haematopoietic cell differentiation, cell signaling, apoptosis, cancer and diseases, and cell proliferation & organ development, and stem cell.

 

                     Apoptosis

Link between miRNAs, growth control and programmed cell death have also come from other species.  The phenotype of mir-48; mir-84; mir-241 mutants in C. elegan is one of cellular overgrowth14.  In D. rerio, the zygotic removal of Dicer results in a larval growth arrest15.  In additional, in M. Musculus, removal of Dicer in the limb mesoderm leads to a dramatic programmed cell death in the developing limb19. 

Drosophila bantam encodes a 21 nucleotide regulatory miRNA that has an anti-apoptotic17 and also regulated growth14.  The sequence of bantam miRNA was found to be partially complementary to the 3’ UTR sequence of its target, the pro-apoptotic gene hid.  Mature miRNAs of bantam inhibit cell death by translational repression of the hid miRNAs.

                  Cancer and Diseases

Many miRNAs are de-regulated in primary human tumors18-19.  Many human miRNAs are located at genomic regions linked to cancer19.  Of particular interest is the mir-17 miRNA cluster, which is in a region on human chromosome 13 that is frequently amplified in B-cell lymphomas18.  Another potential link between miRNAs and human disease comes from the identification of an essential cofactor for the miRNA biogenesis enzyme Drosha.  This cofactor is encoded by DGCR8, which maps to chromosomal region 22q11.2 which is commonly deleted in DiGeorge Syndrom20. 

In case of leukemia, the case of miRNA involvement is more suggestive.  A majority of DNA alterations occur in regions on chromosome 13 that are associated with mantel cell lymphoma and B cell chronic lymphocytic leukemia21.  The miR-15a, miR-16 cluster resides in this region, which is now limited to about 30 kb, and lies between exon 2 and 5 of the LEU2 gene.  LEU2 has been excluded as the tumor suppressor gene21.  Further, miR-15 and miR-16 are frequently down regulated or deleted in CLL which both miRNA genes are acting as tumor suppressor genes will surface. 

                     Cell Proliferation and Organ Development

Many miRNAs have intriguing tissue specific or developmental stage specific expression, suggesting they are involved in developmental processes.  Organ specific miRNAs have been identified in lung, spleen, liver, heart, and kidney22 - 23, suggesting that miRNAs and found that miR-427 is expressed transiently after mid-blastula transition24.  Further, 17 of 24 miRNAs are detected at specific genes of development, and are continuously expressed until tadpole stage.

                  Cell Signaling

miRNA are involved in many aspect of cellular control, including regulation of signaling pathway25-27.  One case of miRNA involvement in cell signaling involves insulin signing.  In a study designed o reveal possible roles of miRNAs in pancreatic endocrine cells, an islet-specific miRNA (miR-375) was identified from a library of small RNA cloned from pancreatic b-cell line MIN6 and pancreatic a-cell line27.  Several other conserved miRNAs other conserved miRNAs were identified but miR-375 is the most abundant miRNA in the pancreatic cell lines MIN6 and TC1.  siRNA silencing of Mtpm showed similar effects as the of miR-375, suggesting it could be a target of miR-37527.  As a new regulator of insulin signaling miR-375 has the potential to become a new pharmacological target for diabetes therapy.  In general, the targets of miRNAs may substantially increase the number of therapeutic targets for those genes involved in disease states. 

                  Control of Developmental Timing

Interest in the genes controlling developmental timing in C. elegans led to the cloning of the first miRNA, line-4, and the identification of the first miRNA target, lin-1428.  The developmental-timing, or heterochronic, pathway regulates stage-specific processes during C. elegans larval development.  For example of the C. elegans life form, only at the adult stage, in line-4 mutant animals, the seam cells (the developmental fate of several stem cells in the lateral hypodermis) repeat the cell division pattern that characterized the first larval stage (L1) and fail to differentiate.  This mutant phenotype has been interpreted as a heterochronic change with the developmental clock being stuck at the L1 stage.  This result indicated that gain-of-function mutations in the line-4 miRNA lead to an identifiable phenotype, whereas loss-of-function mutations in line-14 result in an opposite where the seam cells skip the cell division of the first larval stage25.  Three miRAs of the let-7 family, mir-48, mir-84, and mir-241 are act redundantly to control the next developmental transition, from the second larval stage (L2) to the third larval stage (L3)1, 14.  The miRNA let-7 identified controls the tradition from the fourth larval stage11 to the adult stage.  Those facts bring attend that at least two miRNA families and at least four miRNAs are involved in the control of developmental timing in C. elegans.  As the lin-4 and let-7 miRNA families are conserved, they might play similar roles in other organism. 

                  Haematopoietic Cell Differentiation

There is scientific hypothesis that miRNAs are transcriptional regulated in different cell types such that there is extensive cross-talk between transcription and posttranscriptional regulation and that distinct miRNAs are active.  In the hematopoeitic system, ectopic expression of miR-181, which is highly expressed in thymus and not in most other tissue, increases the fraction of B-lineage cells both in vitro and vivo39.  miRNAs also likely play important role in maintaining mature cell function, as has been described in fat and insulin metabolism4.  miRNAs are especially abundant in the adult brain, suggesting a key role for them in neuronal functional and plsticity14. 

                     Stem Cell

The complex and cellular diversity of the adult brain arises from the proliferation and differentiation of small number of stem cells.  This intrinsic state of stem cells depends on their spatial and temporal history and affects their responsiveness to extrinsic signals from the microevironment.  Stem cell self-renewal and different along neural and glial lineages are defined by the dynamic interplay between transcription, epigenetic control, and posttranscriptional regulators, including miRNAs.  Given the involvement in developmental processes of the few documentation miRNAs, it is not surprising that a number of miRNA genes are preferentially expressed in stem cells, and maybe involved in maintaining the pluripotent state30. 

miRNAs are especially attractive candidates for regulating stem cell elf-renewal and cell fate decisions, as their ability to simultaneously regulate many target privies a means for coordinated control of concerted gene action.  Although direct evidence for a functional role for miRNAs in stem cell biology is just emerging, tantalizing hints regarding their involvement based on expression patterns, predicted targets, and over-expression studies suggest that they will be key regulators.  For example, in neuron stem cells, miRNAs has been shown by the rescue of brain morphogenesis in maternal-zygotic dicer zebra fish mutants by injection of miR-43012.  This demonstrates that an individual miRNA can trigger large scale changes in development, perhaps as a result of global changes in the transcriptiome14.  miRNAs may also define regulatory patterning in the developing central nervous system.  miRNAs are likely important regulatory for stem cell self-renewal as well as a stem cell differentiate.  They down-regulate stem cell maintenance genes and activate lineage-specific genes.  These transitions require a raid switch in gene expression profiles.

Reference

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