microRNA

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Hairpin structure of a pri-microRNA

microRNA ( Greek micros 'small'), abbreviated miRNA or miR , are short, highly conserved, non-coding RNAs that play an important role in the complex network of gene regulation , especially in gene silencing . MicroRNAs regulate gene expression in a highly specific manner on the post-transcriptional level. Generally, microRNAs are between 21 and 23 nucleotides (nt) in size , but they can be several hundred (see figure).

Mechanism of gene regulation

In animals, gene regulation takes place by binding the microRNAs to the 3 ' untranslated region (3'-UTR) of the mRNA of target genes, which, depending on the complementarity of the binding sequence and the proteins involved, are either inhibited in translation or broken down by cutting. Partial complementarity leads to translation inhibition, while perfect base pairing leads to degradation of the target mRNA. While it has long been assumed that translation inhibition dominates of these two mechanisms, more recent studies have shown that the degradation of target mRNA is relatively responsible for a larger proportion of the inhibition of protein production.

history

microRNAs were first described in 1993, but the name microRNA was not coined until 2001. In the nematode Caenorhabditis elegans, the lin-4 gene surprisingly did not code for a protein, but for two small RNA molecules with a length of approximately 60  nt and approximately 20 nt. As far as we know today, the longer molecule is the pre-microRNA. The molecule with approx. 20 nucleotides is the mature microRNA. The smaller RNA lin-4 regulates the lin-14 gene by binding complementarily to the 3'-UTR of the lin-14 mRNA through base pairing and reducing the translation of the mRNA. With the discovery of another miRNA (let-7) it could be shown that this is probably not a rare phenomenon in C. elegans . Let-7 regulates the lin-41 gene. Since let-7 and lin-41 are evolutionarily conserved genes, it became clear that the mechanism of microRNA regulation could also be applied to other multicellular organisms. In recent years, the knowledge about microRNAs has grown steadily. The miRBase database shows an increase of over 4000 sequences in the last two years. The Pubmed database also recorded a sharp increase in publications on the subject of microRNAs. The biological functions of most microRNAs are still unknown. According to computer-based predictions, around 20–30% of the genes in the human genome could be regulated by microRNAs. It can be assumed that several hundred to a few thousand different microRNAs are encoded.

biogenesis

The transcription and effects are described using the example of mammals. With deviations, e.g. B. in the protein components involved, the mechanism of action in the different species is comparable. The genes for the miRNAs are located in the genome; they are transcribed by an RNA polymerase II or III. The resulting primary transcript has a length of 500 to 3000 nucleotides and has the usual poly-A tail at the 3 'end and a 7-methylguanosine cap at the 5' end. The primary transcript is called primary microRNA (pri-miRNA) and forms a loop. The RNase III ( Drosha ) and the dsRNA binding protein DGCR8 (corresponds to Pasha in Drosophila melanogaster ) form a microprocessor complex through which the pri-miRNA in the nucleus of a cell is transformed into a precursor microRNA (pre-miRNA) about 70-80 nucleotides in size. is litigated. The pre-miRNA forms a characteristic hairpin structure . The pre-miRNA is actively exported into the cytoplasm via the nuclear pores of the nuclear membrane by Exportin-5 in the presence of Ran-GTP as a cofactor . In the cytoplasm, the pre-miRNAs are cut into 17–24 nt long ds-miRNAs by the RNAse III enzyme Dicer . Dicer interacts with the ds-RNA binding protein TRBP (RDE-4 in C. elegans and Loquacious in Drosophila melanogaster ), causing the miRNA duplex to unravel and become single-stranded. It starts at the end with the lower thermodynamic stability. Depending on this, the miRNA strand with the 5 'terminus at its end forms the mature miRNA, which is also called guide RNA. The opposite strand is marked with an asterisk in annotation. This miRNA * can possibly also have a regulatory effect in a few cases. The mature miRNA is incorporated into a ribonucleoprotein complex (miRNP), which is very similar to the RISC complex of the RNAi pathway. With this miRISC complex, the activity of the target genes can be downregulated by two methods. This depends on the degree of complementarity between the microRNA and the mRNA of the target gene, as well as on the RNA binding proteins of the Argonaut family . If the binding sequence partially matches, translation is inhibited by binding. If the complementarity is high, the target mRNA is cut. The mechanisms of action of the microRNAs and the so-called small interfering RNAs ( siRNAs ) show clear parallels. siRNAs are processed by the RNase III Dicer from long double-stranded RNAs (dsRNAs) and incorporated into the siRISC complex as 21–28 nt single-stranded RNAs. This complex mediates the mRNA degradation.

RNA interference

A breakthrough in science is the discovery that artificially induced double-stranded RNA in C. elegans also leads to an efficient and specific gene knockdown . This mechanism is known as RNA interference (RNAi). The two US scientists Andrew Z. Fire and Craig C. Mello received the Nobel Prize in Physiology or Medicine in 2006 for their discovery of the mechanism of RNA interference .

Expression

MicroRNAs have very different expression patterns and are regulated differently in development and in physiological processes. The expression is mostly organized in a tissue-specific manner together with nearby genes. MicroRNAs can be located within exons or introns of protein-coding genes, or in non-coding regions. Some microRNAs have their own promoters . A few microRNAs are present in a cluster and are transcribed together .

Current research

The study of microRNA is a new and very topical topic in molecular and cell biology. It is more difficult to work with miRNAs than with mRNAs , as they are quickly degraded due to their small size and ubiquitous occurrence of degrading enzymes. The quantification of miRNA therefore requires constant work under cooling and specially prepared, RNase- free equipment. Classically, miRNA expression levels are investigated by transcribing the RNA into synthetic DNA (cDNA) and then quantifying it using quantitative PCR . It has recently become possible to measure miRNA expression on special microarray platforms, a method that is enjoying growing popularity. Although this allows the simultaneous inventory of hundreds of miRNAs, the post-processing of the data, as is usual with microarrays, is complex and often associated with large scatter. However, this can often be compensated for by the subsequent analysis of the data sets, which in the best case also contain information on the regulated mRNAs.

In recent years it has been found that miRNAs function as important regulators of gene translation ( translation ) after gene overwriting (transcription). This takes place via the specific attachment to mRNA molecules, the translation of which into proteins is thus made difficult, completely prevented or even facilitated.

So far, over 800 different miRNAs have been detected in mammalian cells; In humans, over 1,800 different miRNA species are currently known, which are available in collections, so-called libraries (as of 11/2013,) The comparison of invertebrate and vertebrate cells shows that the structure of some of these molecules is highly conserved, which suggests a suggests important common evolutionary function in very different species.

Experimental and computer science studies suggest that every miRNA can regulate some mRNA molecules, and that 20–30% of all human genes are controlled by miRNAs.

The type and number of miRNA molecules produced in the cell nucleus often shows a close correlation with the level of development of the cell (cell division, differentiation into certain cell types, apoptosis (programmed cell death in the event of errors)). The miRNAs work together with transcription factors (TFn) in a regulatory network . Current studies confirm the critical function of miRNAs in the early development processes of animals, for example neurogenesis , myogenesis (muscle formation), cardiogenesis ( heart formation) and hematopoiesis (blood formation). miRNAs also play an important role in plants.

Furthermore, it has been suggested that miRNAs are very important for the suppression of cellular transformations such as tumor formation , since an incorrect formation of miRNA molecules in cells intensifies these undesired processes. Deregulation of miRNAs has been observed in various tumor types and appears to have tumor-specific characteristics. The influence of the p53 protein, known as a tumor suppressor, on the maturation of various miRNAs that inhibit cell growth also suggests this conclusion.

Recent research shows that certain miRNAs are important for the maintenance of pluripotency and self-renewal of embryonic stem cells . In the future, microRNAs could therefore be useful molecular biological tools for manipulating stem cells.

For research purposes, cellular microRNA molecules can be inhibited with the aid of complementary antagomires .

The latest results indicate that miRNA is absorbed through food and influences processes in the body.

The base sequences of the various miRNAs are identical or almost identical in mammals. These matches can be checked on the Internet using database queries (e.g. miRBase). At the same time, there is an organ specificity in all species, so that in the same organs of different mammals identical miRNA types take over their functions within the framework of the modulation of protein biosynthesis.

Important microRNAs in the human cell

The microRNA-335 (-5p), whose transcript template is in the gene sequence for MEST , was recognized as an important regulator in the development of malignant tumors, but also recognized as a cancer-promoting Oncomir .

The miR-193b is regulated by STAT5 and modulates the expression of KIT , a receptor tyrosine kinase that is important for blood stem cells . It is involved in the renewal and expansion of blood stem cells and progenitor cells (blood precursor cells). Dysregulation of miR-193b is linked to the pathogenesis and aggressiveness of acute myeloid leukemia (AML).

The microRNA-145 reduces the expression of Oct-4 , a gene typical of stem cells. Their failure therefore promotes the development of cancer stem cells.

The miR-302 family microRNAs are typical of human embryonic stem cells and are regulated by the pluripotency genes Oct-4 and Sox-2 .

Nomenclature rules

The naming of the microRNAs is based on the chronological order of sequencing. The first three letters designate the organism (e.g. hsa- Homo sapiens ).

Different precursor sequences and genomic loci expressing identical mature sequences are given names of the form hsa-mir-121-1 and hsa-mir-121-2. Lettered suffixes denote closely related mature sequences - for example, hsa-miR-121a and miR-hsa-121b.

miRNA cloning studies sometimes identify two sequences approximately 22 bases long that are derived from the same predicted precursor. When the relative amounts clearly indicate which is the predominant miRNA form, the mature sequences are assigned by names of the form miR-56 (major product) and miR-56 * (from the opposite arm of the precursor). If the data is insufficient to determine which sequence is the predominant one, names such as miR-142-5p (from the 5 'arm) and miR-142-3p (from the 3' arm) are given.

For example, an older convention that is sometimes used is: B. miR-142 and miR-s-142-AS.

See also

literature

Web links

Individual evidence

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