Reprinted please specify: Spiral · Clinician Research Growth Platform
It is not unreasonable that lncRNA is so red now. It has propped up half the sky in the field of cell life with its powerful and unique regulatory function. In recent years, research on its downstream mechanisms has emerged in an endless stream. Although the downstream regulation mechanism of lncRNA is intricate, it usually cannot be separated from the five levels of gene, transcription, post-transcription, translation and post-translation.
In terms of gene level, there is an inextricable relationship between lncRNA and DNA methylation. This model is commonly associated with the binding of lncRNA to methyltransferase (DNMT1, 3, etc.) and localizes the enzyme to the promoter of the gene (CpG island) and methylation, thereby inhibiting gene transcription.
In addition, lncRNA located in the nucleus can directly bind to DNA sequences to inhibit the transcription process; or bind transcription factors, RNA polymerase complexes, and through histone modifications to affect the transcription process.
Moreover, lncRNA can not only show its strength in the nucleus, but also because it is free to shuttle this feature inside and outside the nucleus, it is also sharp in the cytoplasm. Not to mention the familiar ceRNA, lncRNA can also affect the physiological function of cells through the variable shear, localization and stability of mRNA.
In addition, lncRNA also promotes pri-miRNA cleavage, and sometimes even itself as a precursor of miRNA , after being cleaved into miRNA, to inhibit the expression level of target gene mRNA. As the saying goes, the technology does not press, the lncRNA that is open all the way also intervenes in the protein translation process, either by binding to the mRNA 5'UTR to promote translation; either by binding to a specific protein, targeting mRNA, inhibiting translation; or relying on sORF translation of the polypeptide From production and sales.
LncRNA is so versatile and it makes the protein itch. The two hit it off, and the phosphorylation and localization of the protein were carried out in an orderly manner under the influence of lncRNA.
Although all of the above are the 18 martial arts of lncRNA function, it is still necessary to remember that greedy chews are not bad. The little friends only need to choose the one based on the subcellular localization of lncRNA (related to the regulation function). Will gain something.
a. In the nucleus, first consider whether the gene expression in the nearby 1 million bp has an effect, and some are cis-cis-type; conversely, for the trans-trans-regulated gene, the protein that binds to lncRNA can be screened according to RNA pulldown ;
b. Locate the cytoplasm. If the RNA is bound, the ceRNA is preferred. If the protein is bound, the mechanism of variable shear, stability, gene translation and protein modification can be considered.
As for the overall study of the molecular mechanism of lncRNA, there are always two main strategies to implement.
a. Starting with non-coding RNAs, this is a routine routine for non-coding RNA studies. Starting from different stimuli or treated transcriptomes or expression groups, functional candidate RNA molecules are screened by differential fold and significance, and genomic localization information of non-coding RNA; secondly through positive and negative functions and cell-animal experiments. verification.
The strategy is stable and reliable, and the risk is small. The difficulty lies in the research of the late molecular mechanism. If only the star pathway and related proteins are involved, the indirect molecular mechanism of lncRNA is discussed. However, in order to extract the article, it is necessary to RNA-pulldown, RIP, ChIRP and other experimental techniques to determine lncRNA interacting molecules and binding sites to explore the direct molecular mechanism of lncRNA.
b. Starting from a certain mode of molecular action, it is exactly the opposite. Target an important protein molecule, such as a signal transduction molecule, an enzyme or a transcription factor, or a cell substructure, such as mitochondria, exosomes, etc., by RIP-seq or RNA-seq to detect binding or inclusion. The RNA is screened for candidate RNA molecules according to the enrichment factor and significance, and the functional RNA is subsequently screened by siRNA or high expression.
This strategy has a clear functional molecular mechanism from the beginning of the project design, which is convenient to carry out in the subsequent molecular mechanism research; but the difficulty is how to do the effective separation of RIP-seq and cell substructure in the early stage, which is the reliability of subsequent experiments. And an important guarantee of feasibility.
The two strategies overlap in experimental techniques, but they also have their own unique experimental technical requirements or data analysis strategies. Different strategies are adapted to different topics and laboratory backgrounds, and can be selected based on the characteristics of the project and the laboratory technology system. Of course, the two strategies can also be applied at the same time, complement each other and testify against each other to achieve better verification results.