CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators
Abstract Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m6A and m5C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high-throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed <jats:underline>C</jats:underline>RISPR <jats:underline>i</jats:underline>ntegrated <jats:underline>g</jats:underline>RNA <jats:underline>a</jats:underline>nd <jats:underline>r</jats:underline>eporter sequencing (CIGAR-seq). Using CIGAR-seq, we discovered NSUN6 as a novel mRNA m5C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on non-overlapping subsets of mRNA m5C sites, and together contributed to almost all the m5C modification in mRNA. Finally, using m1A as an example, we demonstrated that CIGAR-seq can be easily adapted for identifying regulators of other mRNA modification..
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Preprint| Erscheinungsjahr: |
2020 |
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| Erschienen: |
2020 |
| Enthalten in: |
bioRxiv.org - (2020) vom: 05. Okt. Zur Gesamtaufnahme - year:2020 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Fang, Liang [VerfasserIn] |
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| Links: |
Volltext [kostenfrei] |
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| doi: |
10.1101/2020.10.03.324715 |
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| PPN (Katalog-ID): |
XBI018884156 |
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| 520 | |a Abstract Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m6A and m5C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high-throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed <jats:underline>C</jats:underline>RISPR <jats:underline>i</jats:underline>ntegrated <jats:underline>g</jats:underline>RNA <jats:underline>a</jats:underline>nd <jats:underline>r</jats:underline>eporter sequencing (CIGAR-seq). Using CIGAR-seq, we discovered NSUN6 as a novel mRNA m5C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on non-overlapping subsets of mRNA m5C sites, and together contributed to almost all the m5C modification in mRNA. Finally, using m1A as an example, we demonstrated that CIGAR-seq can be easily adapted for identifying regulators of other mRNA modification. | ||
| 700 | 1 | |a Wang, Wen |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Guipeng |e verfasserin |4 aut | |
| 700 | 1 | |a Zhang, Li |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Jun |e verfasserin |4 aut | |
| 700 | 1 | |a Gan, Diwen |e verfasserin |4 aut | |
| 700 | 1 | |a Yang, Jiao |e verfasserin |4 aut | |
| 700 | 1 | |a Tang, Yisen |e verfasserin |4 aut | |
| 700 | 1 | |a Ding, Zewen |e verfasserin |4 aut | |
| 700 | 1 | |a Zhang, Min |e verfasserin |4 aut | |
| 700 | 1 | |a Zhang, Wenhao |e verfasserin |4 aut | |
| 700 | 1 | |a Deng, Daqi |e verfasserin |4 aut | |
| 700 | 1 | |a Song, Zhengyu |e verfasserin |4 aut | |
| 700 | 1 | |a Zhu, Qionghua |e verfasserin |4 aut | |
| 700 | 1 | |a Cui, Huanhuan |e verfasserin |4 aut | |
| 700 | 1 | |a Hu, Yuhui |e verfasserin |4 aut | |
| 700 | 1 | |a Chen, Wei |e verfasserin |4 aut | |
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