Transcriptome analysis of hypoxic cancer cells uncovers intron retention in EIF2B5 as a mechanism to inhibit translation.
Transcriptome analysis of hypoxic cancer cells uncovers intron retention in EIF2B5 as a mechanism to inhibit translation.
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DOI：
10.1371/journal.pbio.2002623
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发表时间：
2017-09
期刊：
影响因子：
9.8
通讯作者：
Koumenis C
中科院分区：
文献类型：
--
作者：
Brady LK;Wang H;Radens CM;Bi Y;Radovich M;Maity A;Ivan C;Ivan M;Barash Y;Koumenis C
细胞通过下调包括翻译在内的能量消耗过程来适应肿瘤微环境中的缺氧应激。为了描述细胞对缺氧的适应机制，我们对常氧和缺氧头颈癌细胞进行了RNA-Seq。这些数据揭示了已知调节RNA加工和剪接的基因的显著下调。外显子水平分析将> 1，000个mRNA分类为缺氧条件下的选择性剪接，并在翻译起始的主要调节因子EIF 2B 5中发现了独特的保留内含子（RI）。值得注意的是，该内含子在实体瘤中以阶段依赖性方式表达。我们研究了这种RI的生物学后果，并证明其包含产生了一个提前终止密码子（PTC），这导致了一个65 kDa的截短蛋白异构体，该异构体对抗全长eIF 2B ε以抑制整体翻译。此外，65 kDa eIF 2B ε的表达导致头颈癌细胞在缺氧条件下的存活增加，这提供了这种亚型使细胞能够适应低氧条件的证据。进一步的工作，以揭示-顺式和-反式调节EIF 2B 5剪接确定了几个因素，影响内含子保留在EIF 2B 5：一个弱的剪接潜力在RI，缺氧诱导的表达和结合的剪接因子SRSF 3，并增加结合总的和磷酸-Ser 2 RNA聚合酶II特异性保留在内含子缺氧。总之，这些数据揭示了差异剪接作为一个以前未表征的模式下缺氧的翻译控制，并支持一个模型，其中缺氧诱导的变化，共转录处理导致选择性保留的PTC含内含子在EIF 2B 5。肿瘤缺氧是许多实体癌的负性预后因素。细胞对缺氧的适应在很大程度上是由基因表达的广泛变化介导的，并使癌细胞能够调整和存活。最近，选择性剪接已牵连在这一过程中。为了鉴定生物学上有影响力的缺氧反应亚型，我们采取了一种无偏倚的方法对常氧和缺氧头颈癌细胞的RNA进行深度测序。该分析鉴定了> 1，000种mRNA在缺氧下选择性剪接，包括参与适应缺氧的选择性剪接基因的显著富集。最值得注意的是，我们在翻译起始因子EIF 2B 5中发现了一个独特的保留内含子，它产生了一个提前终止密码子。我们发现，这种保留的内含子导致65 kDa的截短同种型，其对抗全长eIF 2B ε以抑制整体翻译并增强头颈癌细胞在缺氧下的存活。引人注目的是，这个内含子和几个额外的缺氧诱导的保留内含子在实体瘤中相对于正常组织过表达。从机理上讲，我们认为内含子在缺氧条件下的保留受到弱3′剪接位点RNA聚合酶II（RNAPII）活性变化的影响，并对EIF 2B 5中保留的内含子进行了实验验证，以研究内含子在缺氧条件下的保留。
Cells adjust to hypoxic stress within the tumor microenvironment by downregulating energy-consuming processes including translation. To delineate mechanisms of cellular adaptation to hypoxia, we performed RNA-Seq of normoxic and hypoxic head and neck cancer cells. These data revealed a significant down regulation of genes known to regulate RNA processing and splicing. Exon-level analyses classified > 1,000 mRNAs as alternatively spliced under hypoxia and uncovered a unique retained intron (RI) in the master regulator of translation initiation, EIF2B5. Notably, this intron was expressed in solid tumors in a stage-dependent manner. We investigated the biological consequence of this RI and demonstrate that its inclusion creates a premature termination codon (PTC), that leads to a 65kDa truncated protein isoform that opposes full-length eIF2Bε to inhibit global translation. Furthermore, expression of 65kDa eIF2Bε led to increased survival of head and neck cancer cells under hypoxia, providing evidence that this isoform enables cells to adapt to conditions of low oxygen. Additional work to uncover -cis and -trans regulators of EIF2B5 splicing identified several factors that influence intron retention in EIF2B5: a weak splicing potential at the RI, hypoxia-induced expression and binding of the splicing factor SRSF3, and increased binding of total and phospho-Ser2 RNA polymerase II specifically at the intron retained under hypoxia. Altogether, these data reveal differential splicing as a previously uncharacterized mode of translational control under hypoxia and are supported by a model in which hypoxia-induced changes to cotranscriptional processing lead to selective retention of a PTC-containing intron in EIF2B5. Tumor hypoxia is a negative prognostic factor for many solid cancers. Cellular adaptation to hypoxia is largely mediated by widespread changes in gene expression and enables cancer cells to adjust and survive. Recently, alternative splicing has been implicated in this process. To identify biologically impactful hypoxia-responsive isoforms, we took an unbiased approach to deeply sequence RNA of normoxic and hypoxic head and neck cancer cells. This analysis identified >1,000 mRNAs as alternatively spliced under hypoxia, including a significant enrichment of alternatively spliced genes involved in adaptation to hypoxia. Most notably, we discovered a unique retained intron in the translation initiation factor EIF2B5 that creates a premature termination codon. We show that this retained intron leads to a 65kDa truncated isoform that opposes full-length eIF2Bε to inhibit global translation and enhances survival of head and neck cancer cells under hypoxia. Strikingly, this intron and several additional hypoxia-induced retained introns were overexpressed in solid tumors relative to normal tissues. Mechanistically, we propose that intron retention under hypoxia is influenced by changes to RNA polymerase II (RNAPII) activity at weak 3′ splice sites and carry out experimental validation for the retained intron in EIF2B5 to investigate intron retention under hypoxia.
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DOI：
10.1158/1541-7786.mcr-09-0502
发表时间：
2010-03
期刊：
Molecular cancer research : MCR
影响因子：
--
作者：
Gardner LB
通讯作者：
Gardner LB
影响因子：
7.7
作者：
Choudhry, Hani;Schoedel, Johannes;Oikonomopoulos, Spyros;Camps, Carme;Grampp, Steffen;Harris, Adrian L.;Ratcliffe, Peter J.;Ragoussis, Jiannis;Mole, David R.
通讯作者：
Mole, David R.
影响因子：
5.3
作者：
Galban, Stefanie;Kuwano, Yuki;Gorospe, Myriam
通讯作者：
Gorospe, Myriam
影响因子：
7
作者：
Braunschweig U;Barbosa-Morais NL;Pan Q;Nachman EN;Alipanahi B;Gonatopoulos-Pournatzis T;Frey B;Irimia M;Blencowe BJ
通讯作者：
Blencowe BJ
DOI：
10.1073/pnas.0506139102
发表时间：
2005-09-06
影响因子：
11.1
作者：
Baek, D;Green, P
通讯作者：
Green, P