RNA Interaction
Human microRNAs were comprehensively analyzed
Matrix RNAs transmit information from genes to proteins, while microRNAs play a key role in regulating the work of genes. Scientists from MIPT and the Medical-Genetic Research Center described the complexity of the interaction of microRNAs with matrix and other human RNAs. The work was published in the journal Frontiers in Genetics. (Plotnikova et al., Comprehensive Analysis of Human microRNA–mRNA Interactome).
What are microRNAs and Argonaut proteins?
Ribonucleic Acid (RNA) – one of the main types of molecules that implements genetic information in the cell. It is customary to isolate transport, ribosomal and matrix RNAs. Matrix RNA (mRNA) – an intermediary between the gene storage, deoxyribonucleic acid (DNA), and the protein molecules formed "from genes". Formed on the DNA matrix in in the cell nucleus, mRNA moves into the cytoplasm and, in turn, becomes the matrix on which protein synthesis takes place. However, not all the RNA molecules synthesized by the cell produce proteins, but approximately only 2%. For example, in addition to matrix RNA, the cell produces microRNA with a length of 18-25 nucleotides, on the matrix of which the protein is not synthesized. They will be discussed further.
In cells, microRNAs work in complex with proteins of the Argonauts family (AGO). Such a small microRNA-AGO complex connects to the mRNA in one of its parts. Which mRNA and in which part of it to contact determines the microRNA (~ 2.5 thousand are known for humans). The AGO protein either simply blocks the production of protein with mRNA, or completely destroys the mRNA by "cutting" it. Thus, if the microRNA-AGO complex interacts with certain mRNAs paired for it, then the protein can no longer be synthesized from it. In this case, it turns out that the genes that encoded this mRNA are "silenced", that is, the microRNA, having "captured" the mRNA, affected the work of the genes.
Therefore, despite the fact that microRNA interacts directly with mRNA, this interaction also means the interaction between microRNA and the gene encoding this mRNA. Such "silencing" is one of the many mechanisms regulating gene expression. The regulation of gene expression refers to cellular mechanisms that allow you to control the performance of a particular gene: completely or partially turn off or turn on its work. Improper regulation of gene expression due to a "breakdown" in the functioning of microRNAs can lead to pathologies, including the development of a cancerous tumor.
The understanding of the interactions between mRNA and microRNA is far from complete at the moment. For humans, ~20 thousand mRNAs and 2.5 thousand microRNAs are now known. However, there is no clear understanding of which of them connects with whom. In their previous work, the researchers showed that computer programs designed to predict the interactions of microRNA and mRNA do not work in the best way.
In the new work, the scientists decided to combine experimental data on the number of mRNAs and microRNAs formed in a cell with data on the interaction between them for two types of human cells. Using this data as an example, they examined how the amount of a particular microRNA in a cell is related to how many paired mRNAs are produced in the same cell. It can be assumed that the more microRNAs are formed, the more compounds it should form. It turned out that this was not the case. In addition, the researchers figured out how many pairs are formed and how, that is, with the same or different microRNAs. Scientifically speaking, geneticists have investigated how the expression level and binding activity for microRNA and mRNA are related. They also found out how the behavior of such pairs depends on the type of cells.
Olga Plotnikova, one of the authors of the work, a graduate student at MIPT, says:
"Our research is devoted to studying the interactions of microRNAs and genes. microRNAs are known to be important non–coding small RNAs that regulate gene expression. Earlier we published an article where we showed that the programs used to predict the interactions of microRNAs and genes do not work very well. Therefore, we wanted to get a complete picture of the interactions of microRNAs: who, with whom and how. To do this, we analyzed two currently unique scientific papers with experimental data on the complete interaction between microRNAs and genes in two different human cell lines. Then we correlated these data with the results of other experimental studies, where the level of expression of microRNA and mRNA in the same cell lines was determined. We have shown that not all genes are actively regulated by microRNA, and the potential of microRNA regulation does not directly depend on the level of its expression. We were also able to compare how the microRNA interactions in the two cell lines differ."
Methods
The main problem of experimental study of microRNA interactions is the limitation of methods. One group of methods allows one experiment to test one interaction (the so–called Reporter assay), the other group - to identify all the sites of binding to microRNA, but it does not give information about which microRNA was bound at this point (the so-called CLIP methods). In the CLIP method, what is connected to the AGO protein is fixed and "pulled" by this protein for further recognition of the mRNA interacting with it. Thus, it is possible to identify all the sites of microRNA – mRNA binding, but at the same time not to know which of the thousands of microRNAs has acted.
Recently, two similar techniques have been developed (the CLASH and CLEAR-CLIP methods), which are advanced CLIP technologies. These methods are very complex and are currently used only on two human cancer cell lines: kidneys and liver. In this work, we also used data on the number of mRNAs and microRNAs formed in each of the mentioned cell lines (expression data). To identify the regions of mRNA where interaction with microRNA exactly occurs, additionally, scientists used experimental data from 79 CLIP experiments, which do not contain information about which microRNA interacts, but allow us to confirm that there is interaction with microRNA in this place.
Research results
In silico, scientists have proved that the data of the complete interaction of microRNAs and genes obtained by improved CLIP methods are similar in two different human cell lines, and that they can be compared. It has been shown that most of the microRNA–mRNA complexes are formed by a small amount of mRNA and microRNA. For example, only 1-2% of coding genes form more than ten different interactions. Interesting mRNAs were also identified that exhibit a "spongy effect" – such mRNAs were bound in different parts of the mRNA with a large number of microRNAs (>50). In addition, the researchers were able to identify a group of microRNAs that, on the one hand, are poorly expressed, and on the other, have many interactions. This result is not obvious: it seems that the more strongly a particular microRNA is expressed, the more it will connect with various mRNAs.
Another part of the scientific work was devoted to creating a collection of reliable microRNA-binding regions, that is, places where mRNA and microRNA exactly interact. The online program created on its basis, available by link, analyzes whether the position of interest in the genome of a certain person is located at the site of binding to microRNA. So the program allows you to identify a violation of binding to microRNA and a violation of gene regulation, and therefore a possible cause of hereditary diseases. In the future, it can be used, for example, in the analysis of the genome of patients.
Mapping the full interaction of microRNAs and human genes can help in deciphering the molecular basis of hereditary and acquired diseases.
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