Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing.
Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct long-read sequencing.
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DOI：
10.1038/s41467-023-35858-w
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发表时间：
2023-01-19
影响因子：
16.6
通讯作者：
Rouhanifard, Sara H.
中科院分区：
文献类型：
--
作者：
Tavakoli, Sepideh;Nabizadeh, Mohammad;Makhamreh, Amr;Gamper, Howard;McCormick, Caroline A.;Rezapour, Neda K.;Hou, Ya-Ming;Wanunu, Meni;Rouhanifard, Sara H.
在这里，我们开发并应用了一种半定量方法，通过直接长读纳米孔测序来高置信度地鉴定哺乳动物mRNA上的假尿苷化位点。无修饰转录组的比较分析表明，覆盖深度和特定的k-mer序列是准确碱基判定的关键参数。通过调整这些参数的高置信度的U-to-C碱基调用错误，我们确定了许多已知的假尿苷化位点，并发现以前未报道的尿苷修饰的网站，其中许多落在k聚体是已知的假尿苷酶的目标。使用在中心位置具有单个假尿苷的1000聚体合成RNA对照验证鉴定的位点，证明使用我们的方法的系统性欠调用。我们鉴定了具有多达7个独特修饰位点的mRNA。我们的工作流程允许直接检测来自纳米孔测序数据的天然RNA分子上的低、中和高占用假尿苷修饰以及同一链上的多个修饰。假尿苷（psi）是一种RNA修饰，可以影响其生理学，包括增加半衰期。在本文中，作者使用直接RNA测序鉴定了人类转录组中psi修饰的位点，并提供了psi位点、高psi占用位点和可能在多个位点修饰的转录物的“地面实况”列表。
Here, we develop and apply a semi-quantitative method for the high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identify many known sites of pseudouridylation and uncover previously unreported uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites are validated using 1000-mer synthetic RNA controls bearing a single pseudouridine in the center position, demonstrating systematic under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our workflow allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data and multiple modifications on the same strand. Pseudouridine (psi) is an RNA modification that can affect its physiology, including increased half-life. Here the authors identify sites of psi modification in the human transcriptome using direct RNA sequencing and provide a “ground truth” list of psi sites, sites of high psi occupancy, and transcripts that may be modified at multiple sites.
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影响因子：
14.9
作者：
Hunter S;Jones P;Mitchell A;Apweiler R;Attwood TK;Bateman A;Bernard T;Binns D;Bork P;Burge S;de Castro E;Coggill P;Corbett M;Das U;Daugherty L;Duquenne L;Finn RD;Fraser M;Gough J;Haft D;Hulo N;Kahn D;Kelly E;Letunic I;Lonsdale D;Lopez R;Madera M;Maslen J;McAnulla C;McDowall J;McMenamin C;Mi H;Mutowo-Muellenet P;Mulder N;Natale D;Orengo C;Pesseat S;Punta M;Quinn AF;Rivoire C;Sangrador-Vegas A;Selengut JD;Sigrist CJ;Scheremetjew M;Tate J;Thimmajanarthanan M;Thomas PD;Wu CH;Yeats C;Yong SY
通讯作者：
Yong SY
影响因子：
14.9
作者：
Kuleshov MV;Jones MR;Rouillard AD;Fernandez NF;Duan Q;Wang Z;Koplev S;Jenkins SL;Jagodnik KM;Lachmann A;McDermott MG;Monteiro CD;Gundersen GW;Ma'ayan A
通讯作者：
Ma'ayan A
影响因子：
16.6
作者：
Liu, Huanle;Begik, Oguzhan;Novoa, Eva Maria
通讯作者：
Novoa, Eva Maria
影响因子：
18.2
作者：
Fleming AM;Mathewson NJ;Howpay Manage SA;Burrows CJ
通讯作者：
Burrows CJ
影响因子：
14.9
作者：
Jenjaroenpun P;Wongsurawat T;Wadley TD;Wassenaar TM;Liu J;Dai Q;Wanchai V;Akel NS;Jamshidi-Parsian A;Franco AT;Boysen G;Jennings ML;Ussery DW;He C;Nookaew I
通讯作者：
Nookaew I