Comprehensive profiling of mRNA splicing indicates that GC content signals altered cassette exon inclusion in Ewing sarcoma.
Comprehensive profiling of mRNA splicing indicates that GC content signals altered cassette exon inclusion in Ewing sarcoma.
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DOI：
10.1093/narcan/zcab052
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发表时间：
2022-03
期刊：
影响因子：
5.1
通讯作者：
Toretsky JA
中科院分区：
文献类型：
--
作者：
Graham GT;Selvanathan SP;Zöllner SK;Stahl E;Shlien A;Caplen NJ;Üren A;Toretsky JA
尤文肉瘤(EWS)是一种小而圆的蓝色细胞肿瘤，是儿童第二常见的骨癌。85%的EWS肿瘤表达融合癌蛋白EWS-FLI1，这是t(11；22)相互易位的产物。先前的工作表明，仅靠转录调控并不能完全描述EWS-FLI1的致癌能力，也不能提供一种有效的手段来对患者肿瘤进行分层。使用EWS细胞系和患者样本的研究表明，EWS-FLI1也破坏了mRNA的生物发生。在这项工作中，我们既描述了在EWS肿瘤样本中异常剪接的mRNA的潜在特征，也描述了其他儿科肿瘤类型中的mRNA剪接事件。在这里，我们还使用短读和长读测序来确定有助于我们在尤文肉瘤中观察到的剪接特征的顺式因子。我们的分析表明，盒式磁带外显子上游的GC含量是EWS中mRNA剪接的一个决定性因素。我们还描述了将EWS肿瘤样本与假定的起源细胞--人骨髓间充质干细胞(hMSC-BM)区分开来的特定剪接事件。最后，我们通过基序丰富鉴定了特定的剪接因子PCBP2、RBMX和SRSF9，并从EWS细胞系的肿瘤样本中证实了这一发现。尤文肉瘤中的RNA剪接是通过首先使用细胞系模型的长读测序构建参考转录组，然后使用肿瘤样本进行每一事件的量化来量化的。我们发现尤文肉瘤中改变的RNA剪接的特征之一是较高的GC含量和较低的3‘剪接位点强度。
Ewing sarcoma (EwS) is a small round blue cell tumor and is the second most frequent pediatric bone cancer. 85% of EwS tumors express the fusion oncoprotein EWS-FLI1, the product of a t(11;22) reciprocal translocation. Prior work has indicated that transcription regulation alone does not fully describe the oncogenic capacity of EWS-FLI1, nor does it provide an effective means to stratify patient tumors. Research using EwS cell lines and patient samples has suggested that EWS-FLI1 also disrupts mRNA biogenesis. In this work we both describe the underlying characteristics of mRNA that are aberrantly spliced in EwS tumor samples as well as catalogue mRNA splicing events across other pediatric tumor types. Here, we also use short- and long-read sequencing to identify cis-factors that contribute to splicing profiles we observe in Ewing sarcoma. Our analysis suggests that GC content upstream of cassette exons is a defining factor of mRNA splicing in EwS. We also describe specific splicing events that discriminate EwS tumor samples from the assumed cell of origin, human mesenchymal stem cells derived from bone marrow (hMSC-BM). Finally, we identify specific splicing factors PCBP2, RBMX, and SRSF9 by motif enrichment and confirm findings from tumor samples in EwS cell lines. RNA splicing in Ewing sarcoma was quantified by first constructing a reference transcriptome using long-read sequencing of cell line models, then using tumor samples for per-event quantification. We find that altered RNA splicing in Ewing sarcoma is characterized by, among other things, higher GC content and lower 3' splice site strength.