No, Da Niu tells you how to study lncRNA methylation

The lncRNA molecule binds to microRNAs through the sponge mechanism to exert biological functions. This ceRNA mechanism has already made everyone feel bored. But the big cow is the big cow. If you introduce methylation, you can easily turn it into a treasure. Even if you can make the ceRNA idea of ​​lncRNA become an article with a score of 10 or more on the instant, you must be as curious as Xiaobian. How did you do it.
RNA methylation has received more and more attention as the latest national natural hotspot. More research has begun to focus on the discussion and research of RNA methylation direction, only in the past two months, 10+ RNA There are as many as 16 articles in methylation. In the 2018 country's natural project, the number of successful RNA methylation is four times that of 2017. Many of the projects began to shift from mRNA to focusing on the important role of methylation of non-coding RNA in the process of disease development. For example, the role and mechanism of m6A methylated lncRNAs in mantle cell lymphoma (Peking University), RNA demethylase FTO-mediated lncRNA-m6A modification on the regulation of hepatoma cell reprogramming and mechanism research (Tongji University) )and many more. Clouds order to Tongji University biological Kang nine red team in February 2018, published in Nucleic Acid Research (Impact Factor 11.561), for example tell us something about how the study lncRNA methylated molecules.

1. linc1281 is essential for mESC (mouse embryonic stem cell) differentiation
Studies have shown that linc1281, which is mainly distributed in the cytoplasm, is indispensable for the differentiation of mESCs cells. Silencing of endogenously expressed linc1281 by shRNA method allows cells to exhibit normal self-renewal characteristics, but the genes characteristic of mESCs, Oct4, Sox2 and Nanog, are not expressed. Changes suggest that linc1281 may be associated with other biological functions. The investigators injected lincRNA-silenced cells into immunodeficient mice. Compared to the control group, linc1281 silenced mouse teratoma growth was significantly smaller in volume and weight than control tumors. HE staining and immunohistochemistry results showed that silencing of linc1281 molecule can effectively block the differentiation process of mESCs. In addition, in vitro studies of the ability of mESCs cells to be divided into mesoderm-derived cardiomyocytes and ectodermal-derived neural cell lines revealed that linc1281 had no significant effect on pluripotency-related marker expression, and significantly reduced mESCs differentiation ability. On the other hand, overexpression of linc1281 can enhance the differentiation ability of mESCs.

2. Intensive study of m6A methylation modification in linc1281
Recent studies have shown that m6A RNA methylation modification can affect the cell fate of mESCs. Many mESCs-specific transcripts will be modified by m6A, which naturally raises questions. Will linc1281, which has been thoroughly studied in the early stage, be m6A methylated? Is the lincRNA molecule so important to mESCs also regulated by m6A methylation? MeRIP-qPCR experiments confirmed that linc1281 did undergo methylation modification, and shRNA silencing down-regulated the RNA methylase Mettl3 to decrease the methylation level of linc1281, demonstrating that Mettl3 is an RNA methylase that regulates linc1281

3. The level of methylation of linc1281 is positively correlated with the biological function of the molecule.
Linc1281 was 1306 nt in length, and there were three m6A motif RRACUs in the whole sequence. The transgenic and deletion mutant plasmids were transfected into HEK293FT cells to express the exogenous linc1281. The experimental results demonstrate that the mutation of the linc1281 motif significantly reduces the methylation level of linc1281. Overexpression of wild-type linc1281, linc1281 and m6A-modified NC mutants with m6A mutations in cells lacking linc1281, RT-PCR showed that the cell line lacking m6A was not significantly different in myocardial cell trend, but instead m6A modified. The cell line with overexpression of linc1281 was most prominent, and the plasmid of sh1281+linc1281 could significantly promote neural differentiation. It can be seen that the methylation level of linc1281 was positively correlated with the differentiation degree of mESCs. Cell-replenishment experiments demonstrated that the stem cell differentiation ability of the linc1281 knockout cell line can be restored only under the overexpression of the m6A motif intact linc1281. It is concluded that the m6A modification level of linc1281 is directly related to stem cell differentiation ability.

4. linc1281 methylation can affect the ceRNA model of the molecule
Since m6A methylation of linc1281 does not affect the expression of linc1281, how does m6A methylation affect the biological function of this molecule? Through software prediction and cell localization experiments, it was found that the let-7 family and linc1281 bind to each other and have a high abundance in the cytoplasm. The luciferase reporter assay revealed that the microRNA mimcs can be reduced by co-transforming the full length of linc1281 and micor RNA mimics. The fluorescence intensity of linc1281, on the contrary, if the linc1281 and microRNA binding regions were mutated so that they did not bind, microRNA mimics could not significantly reduce the fluorescence intensity of linc1281, and the expression of let-7 was negatively correlated with linc1281, proving that let- The 7 family and lin1281 did have direct binding. Combined with the previously constructed plasmid sh1281+linc1281 and sh1281 point mutations, the expression of let-7 was not significantly changed after the point mutation treatment of m6A motif. On the contrary, the expression of let-7 in the linc1281 plasmid was significantly down-regulated, which means that m6A modification The binding of the linc1281 and let-7 family microRNAs, ie, the m6A methylation modification, affects the ceRNA model of the non-coding RNA molecule. The authors further confirmed by silencing, mutation processing and related cell complementation experiments of METTL3 that the m6A modification of linc1281 can indeed affect the binding of linc1281 and microRNA. This rationally explains how m6A affects biological function without affecting expression levels.

Summary :
In the field of RNA methylation research, the methylation of mRNA is currently the most studied, while the methylation of non-coding parts (lncRNA, circRNA, pri-miRNA, etc.) is relatively rare, relatively non-coding RNA. Methylation studies can be said to be more fashionable. The cloud sequence bio- public has previously compiled a summary of the RNA methylation studies of non-coding RNAs (links before the insertion of the public number). We have divided the applicable population of RNA methylation into two categories: 1 For certain mRNA, lncRNA, circRNA has a certain research basis, you can consider the introduction of RNA methylation, detect whether RNA methylation has occurred and whether the modification can directly correlate with the cell phenotype, publish a high quality RNA methylation article. 2 Researchers without previous basic data can also simultaneously perform RNA methylation detection and transcriptome sequencing (mRNA sequencing, lncRNA sequencing, circular RNA sequencing), rapidly selecting RNA methylation and changing transcriptome levels. The genes that seek to establish a relationship between the two groups of studies can also increase the expression of some related enzymes upstream of RNA methylation (methylase, demethylase, etc.).
Focus : Cloud-sequence comprehensive RNA methylation sequencing service is your high-quality choice, non-coding RNA and encoding RNA molecules for methylation sequencing, three kinds of RNA methylation modification (m6A, m5C, m1A), everything you want Send a high score article? what are you waiting for?

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Cloud order related product recommendation:
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Past review :
Cloud Sequence Bio's Latest m6A "RNA Methylation" Research Summary - Non-coding RNA Methylation
Fancy dog ​​abuse: circRNA and m6A love each other
Plant Cell: Arabidopsis Discovers New m6A RNA Demethylation Modification Enzyme
Cancer cell: Professor He Chuan discovered the important function of m6A RNA methylation
Nature Heavy: m6A RNA methylation is involved in the key aspects of hematopoietic stem cell development!
 

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