The gospel of fat paper! Knocking out the Zfp217 gene can take away the "lifebuoy" in your waist through RNA m6A

"By the way, donuts, pearl milk tea instant noodles! Hot pot rice, big dish chicken, take away and take away!" Obesity drives away, health is there, RNA methylation finally starts with fat differentiation. The genes involved in fat formation and differentiation are no secret. It is our ultimate goal to reduce body fat and maintain body shape. How can post-transcriptional modification in fat differentiation be regulated (di)? This time we talked about how the Zfp217 gene knockout "burns your calories" by RNA m6A methylation!
m6A methylation, as an important post-transcriptional modification, plays an important role in various cases and physiological processes, affecting mRNA stability, maturation, variable splicing, altering the translation efficiency of mRNA proteins, affecting miRNA maturation, etc. The presence of "Wanjin Oil" has made the methylation of RNA m6A highly attractive. However, most of the beginnings of RNA m6A start from the enzyme to the termination of the target molecule. These methylation-related enzymes indirectly change the cell phenotype by affecting the level of methylation of downstream targets. But this time is different. The story originated from the important gene Zfp217 (zinc finger protein) related to adipogenic differentiation. It is precisely this highly functional protein that can be directly related to FTO, YTHDF2 and other methylation. Protein interactions, which cause a wide range of changes in the apparent transcriptome, this article was also published in the famous Nucleic Acid Research (11.9 points) magazine, cloud sequence organisms take you to see that this does not directly interfere with methylase How to tell the scientific story of a high-methyl article.
Published issue: Nucleic Acid Research
Impact factor: 11.9
Experimental method: MeRIP-seq RNA-seq MeRIP-qPCR Co-IP ChIP-qPCR
Original link: Zfp217 mediates m6A mRNA methylation to orchestrate transcriptional and post-transcriptional regulation to promote adipogenic differentiation (doi: 10.1093/nar/gkz312)

Figure 1: Zfp217 mediates mRNA m6A modification to regulate transcription and post-transcriptional modification to promote adipogenic differentiation
The shaded part is the highlight of this article (red part of the experimental part, cloud order provides one-stop service)
1. Zfp217 knockout significantly blocked fat differentiation
In 3T3L1 cells, both siRNA and CRISPR/Cas9 knockout Zfp217 showed significant blockade of fat formation, and the expression of related important genes such as PPARγ, P2, LPL, etc. were significantly down-regulated. The absence of Zpf217 clearly caused the formation of fat.

Figure 2: Significant down-regulation of adipose differentiation-related genes after Zfp217 knockout
2. Zfp217 knockout brings a wide range of m6A modification levels
The overall level of m6A modification (the cloud sequence provides this service) demonstrated that the m6A modification has a broad rise in Zfp217 knockout cells, and this increase occurs early in the knockout rather than 2 days after cell induction culture.

Figure 3: Overall level of mRNA m6A detection
3. RNA-seq combined with MeRIP-seq to explore the molecular mechanism of Zfp217
RNA-seq sequencing was performed after Zfp217 knockout (the cloud sequence can provide this service) . Compared with wild type, 1431 genes were up-regulated and 941 genes were down-regulated. GO analysis indicated that most of the down-regulated genes and cell cycle, RNA processing As well as lipid biosynthesis, it is suggested that Zfp217 may be involved in liposome metabolism and RNA processing.

Figure 4: RNA-seq data collation and GO analysis results
Merip-seq/m6A RNA methylation sequencing (the cloud sequence provides this service) can be seen in comparison between control and Zfp217 knockout 3T3L1 cells. The motif analysis is consistent with the classical GGACU motif and the m6A site is more Exist in the 5'UTR and 3'UTR regions, the number of differentially methylated regions is 3332. Since the difference in methylation occurs in 0 days instead of 2 days, it is proved that the change in methylation level is dependent on Zfp217, and Zfp217 knockdown may inhibit cell proliferation and promote apoptosis. Cell cycle-associated genes are derived from current RNA-seq data, but not in the combined analysis of MeRIP-seq and RNA-seq (the cloud sequence provides this service) data, suggesting that the cell cycle is not predominant and Zfp217 knockout After m6A changes the most relevant biological functions.

Figure 5: MeRIP-seq RNA-seq combined analysis
Of course, other key genes, such as Ccdc141 (Up), Efcab11, Hspa1a for qPCR and MeRIP-qPCR (the cloud sequence can provide this service) verification, the verification results are consistent with the sequencing results. The data suggest that Zfp217 is capable of positively regulating important genes involved in the differentiation of adipose differentiation.

Figure 6: MeRIP-qPCR and qPCR verification
4. Highlights: Zfp217 and YTHDF2 interact to maintain FTO demethylase activity
Studies in tumors have shown that Zfp217 can function in combination with other proteins. At this time, IP experiments with labeled overexpressing cell line HEK293T cells are disappointing results of Co-IP (the cloud sequence can provide this service) . The well-known FTO, ALKBH5 and METTL3/4 star methylases were not found, but in 3T3L1, the authors successfully demonstrated the binding of YTHDF2 and Zfp217 by Co-IP. This binding is independent of RNA. The results of cell localization indicated that both proteins were widely distributed outside the nuclear core, but Zfp217 was distributed more extranuclear than the intranuclear part. Laser confocal showed that the colocalization of YTHDF2 and Zfp217 indicated that they could synergistically play biological functions.

Figure 7: Subcellular localization shows that YTHDF2 and Zfp217 are co-localized in fine
Because it has been previously reported that YTHDF2 can maintain the m6A modification level by inhibiting FTO activity under heat stress, as expected, Zfp217 is the key factor to break this balance. Overexpression of Zfp217 can relieve YTHDF2 inhibition of FTO. In turn, the state of the cells is biased toward the demethylation direction. Zfp217 does not bind to RNA, but instead disrupts the localization of YTHDF2 to complement the demethylation ability of FTO.

5. Highlights: Zfp217 can directly act as a transcription factor to activate FTO transcription
Since Zfp217 has eight C2H2 zinc finger domains and a transfer activation domain, combined with biosignal analysis and ChIP-qPCR (the cloud sequence can provide this service) , the authors found that Zfp217 is indeed a transcription factor activity protein that can be combined with FTO. The promoter region regulates the transcriptional activity of the target gene, and FTO is one of the downstream target genes.

Figure 8: FTO TSS upstream combined with Zfp217
Long-term focus on methylases has limited our horizons, so take a look at this article. Research on RNA methylation Nowadays, researchers have been accustomed to artificially interfering with methylation-related Writer, Eraser and Writer to see the cell phenotype, if not directly knocked out or overexpressed these star molecules, No other ideas? This article is the best template. The Co-IP approach to finding proteins that bind directly to these celebrity proteins and look for powerful upstream molecules for research will be another important step in RNA methylation research. direction.
Yunxu Bio provides exclusive one-stop service for RNA methylation sequencing in China
Cloud-sequence organisms provide colorimetric detection of overall m6A methylation modification levels, RNA methylation sequencing, MeRIP-qPCR validation, RIP and RNA Pull Down mechanisms . RNA methylation sequencing technology is the real implementation of m6A, m5C and m1A modification, detection of molecules in addition to mRNA, but also detection of circular RNA, LncRNA and other non-coding RNA. Since 2016, the number of samples has accumulated more than 5000+, and the integrated power of MeRIP has reached more than 98%.
Now, in order to solve the problem of small sample size of customers, ultra-micro-MeRIP sequencing technology is introduced, and 500 ng of total RNA can be used for sequencing experiments. Special samples can also be sequenced for RNA methylation, such as serum, plasma, exosomes, and paraffin samples.
Cloud order related product recommendation:

  • m6A modified overall level detection

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Past review:

  • look here! How do miRNAs get high scores on m6A hotspots?

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  • Occupy C! How does cloud-sequence bioanalysis quickly detect RNA methylation-related enzymes & RNA methylation levels in various cancers (on)

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  • Cloud order customer 12 points top article, teach you how to use RIP sequencing to play the molecular mechanism!

  • Nat. commun | Mettl3-mediated m6A RNA methylation regulates the fate of bone marrow mesenchymal stem cells and osteoporosis

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  • Cloud Sequence Bio's Latest "RNA Methylation" Research Summary - Arabidopsis

  • Nature Breakthrough Research - RNA Methylation Newly Modified m1A Cloud Sequence Creature

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  • Cloud Sequence Bio's latest m6A "RNA methylation" research summary - non-coding RNA articles

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