Guvenek Aysegul, Shin Jihae, De Filippis Lidia, Zheng Dinghai, Wang Wei, Pang Zhiping P, Tian Bin
Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School, Newark, NJ, United States.
Rutgers School of Graduate Studies, Newark, NJ, United States.
Front Genet. 2022 May 18;13:840369. doi: 10.3389/fgene.2022.840369. eCollection 2022.
RNA stability plays an important role in gene expression. Here, using 3' end sequencing of newly made and pre-existing poly(A)+ RNAs, we compare transcript stability in multiple human cell lines, including HEK293T, HepG2, and SH-SY5Y. We show that while mRNA stability is generally conserved across the cell lines, specific transcripts having a high GC content and possibly more stable secondary RNA structures are relatively more stable in SH-SY5Y cells compared to the other 2 cell lines. These features also differentiate stability levels of alternative polyadenylation (APA) 3'UTR isoforms in a cell type-specific manner. Using differentiation of a neural stem cell line as a model, we show that mRNA stability difference could contribute to gene expression changes in neurogenesis and confirm the neuronal identity of SH-SY5Y cells at both gene expression and APA levels. In addition, compared to transcripts using 3'-most exon cleavage/polyadenylation sites (PASs), those using intronic PASs are generally less stable, especially when the PAS-containing intron is large and has a strong 5' splice site, suggesting that intronic polyadenylation mostly plays a negative role in gene expression. Interestingly, the differential mRNA stability among APA isoforms appears to buffer PAS choice in these cell lines. Moreover, we found that several other poly(A)+ RNA species, including promoter-associated long noncoding RNAs and transcripts encoded by the mitochondrial genome, are more stable in SH-SY5Y cells than the other 2 cell lines, further highlighting distinct RNA metabolism in neuronal cells. Together, our results indicate that distinct RNA stability control in neuronal cells may contribute to the gene expression and APA programs that define their cell identity.
RNA稳定性在基因表达中起着重要作用。在此，我们利用新合成的和预先存在的多聚腺苷酸（poly(A)+）RNA的3'端测序技术，比较了多种人类细胞系中的转录本稳定性，包括HEK293T、HepG2和SH-SY5Y。我们发现，虽然mRNA稳定性在各细胞系中总体上是保守的，但与其他两种细胞系相比，具有高GC含量且可能具有更稳定二级RNA结构的特定转录本在SH-SY5Y细胞中相对更稳定。这些特征还以细胞类型特异性的方式区分了可变聚腺苷酸化（APA）3'非翻译区（UTR）异构体的稳定性水平。以神经干细胞系的分化为模型，我们表明mRNA稳定性差异可能导致神经发生过程中的基因表达变化，并在基因表达和APA水平上证实了SH-SY5Y细胞的神经元特性。此外，与使用3'-最末端外显子切割/聚腺苷酸化位点（PASs）的转录本相比，使用内含子PASs的转录本通常稳定性较低，尤其是当含有PAS的内含子较大且具有强5'剪接位点时，这表明内含子聚腺苷酸化在基因表达中大多起负面作用。有趣的是，APA异构体之间的mRNA稳定性差异似乎缓冲了这些细胞系中PAS的选择。此外，我们发现包括启动子相关长链非编码RNA和线粒体基因组编码的转录本在内的其他几种多聚腺苷酸（poly(A)+）RNA在SH-SY5Y细胞中比其他两种细胞系更稳定，进一步突出了神经元细胞中独特的RNA代谢。总之，我们的结果表明，神经元细胞中独特的RNA稳定性控制可能有助于定义其细胞身份的基因表达和APA程序。
