iCLIP identifies novel roles for SAFB1 in regulating RNA processing and neuronal function.
iCLIP identifies novel roles for SAFB1 in regulating RNA processing and neuronal function.
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DOI：
10.1186/s12915-015-0220-7
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发表时间：
2015-12-22
期刊：
影响因子：
5.4
通讯作者：
Uney JB
中科院分区：
文献类型：
--
作者：
Rivers C;Idris J;Scott H;Rogers M;Lee YB;Gaunt J;Phylactou L;Curk T;Campbell C;Ule J;Norman M;Uney JB
SAFB 1是一种RNA结合蛋白，参与多种细胞过程的调节，如转录调节、应激反应、DNA修复和RNA加工。为了进一步了解SAFB 1的功能，我们使用了iCLIP，并在全基因组水平上绘制了它与RNA的相互作用。iCLIP分析发现SAFB 1结合富集，特别是在外显子、ncRNA、3'和5'非翻译区。发现SAFB 1以最高频率识别富含嘌呤的GA阿加基序，因此它可能结合核心阿加、GAA或AAG基序。验证性RT-PCR实验表明，SAFB 1敲除会改变具有SAFB 1交联位点的编码和非编码基因的表达。例如，我们发现神经细胞粘附分子（NCAM 1）和ASTN 2的亚型特异性表达受到SAFB 1的影响，并且来自miR-17-92簇的miR-19 a的加工受到SAFB 1的调节。这些数据表明，SAFB 1可能会影响选择性剪接，并使用NCAM 1小基因，我们表明，SAFB 1敲低改变了三个NCAM 1选择性剪接异构体的表达。然而，当阿加、GAA和AAG基序突变时，SAFB 1敲低不再介导NCAM 1 9-10可变剪接形式的减少。为了进一步研究SAFB 1与剪接的关系，我们使用外显子阵列分析，发现SAFB 1敲低介导了统计学上显著的替代外显子的上调和下调。使用RNA基序进一步分析以研究基序对（阿加，然后是阿加、GAA或AAG）与可变剪接外显子之间的关联频率，发现与下调的外显子存在高度显著的相关性。总之，我们的数据表明SAFB 1将在中枢神经系统调节突触功能中发挥重要的生理作用。我们发现，SAFB 1调节海马神经元树突棘密度，因此提供了支持这一结论的经验证据。iCLIP表明，SAFB 1具有以前未表征的特异性RNA结合特性，有助于协调编码和非编码基因的亚型特异性表达。这些基因调节剪接、轴突和突触功能，并与神经精神疾病相关，表明SAFB 1是关键神经元过程的重要调节因子。本文的在线版本（doi：10.1186/s12915-015-0220-7）包含补充材料，可供授权用户使用。
SAFB1 is a RNA binding protein implicated in the regulation of multiple cellular processes such as the regulation of transcription, stress response, DNA repair and RNA processing. To gain further insight into SAFB1 function we used iCLIP and mapped its interaction with RNA on a genome wide level. iCLIP analysis found SAFB1 binding was enriched, specifically in exons, ncRNAs, 3’ and 5’ untranslated regions. SAFB1 was found to recognise a purine-rich GAAGA motif with the highest frequency and it is therefore likely to bind core AGA, GAA, or AAG motifs. Confirmatory RT-PCR experiments showed that the expression of coding and non-coding genes with SAFB1 cross-link sites was altered by SAFB1 knockdown. For example, we found that the isoform-specific expression of neural cell adhesion molecule (NCAM1) and ASTN2 was influenced by SAFB1 and that the processing of miR-19a from the miR-17-92 cluster was regulated by SAFB1. These data suggest SAFB1 may influence alternative splicing and, using an NCAM1 minigene, we showed that SAFB1 knockdown altered the expression of two of the three NCAM1 alternative spliced isoforms. However, when the AGA, GAA, and AAG motifs were mutated, SAFB1 knockdown no longer mediated a decrease in the NCAM1 9–10 alternative spliced form. To further investigate the association of SAFB1 with splicing we used exon array analysis and found SAFB1 knockdown mediated the statistically significant up- and downregulation of alternative exons. Further analysis using RNAmotifs to investigate the frequency of association between the motif pairs (AGA followed by AGA, GAA or AAG) and alternative spliced exons found there was a highly significant correlation with downregulated exons. Together, our data suggest SAFB1 will play an important physiological role in the central nervous system regulating synaptic function. We found that SAFB1 regulates dendritic spine density in hippocampal neurons and hence provide empirical evidence supporting this conclusion. iCLIP showed that SAFB1 has previously uncharacterised specific RNA binding properties that help coordinate the isoform-specific expression of coding and non-coding genes. These genes regulate splicing, axonal and synaptic function, and are associated with neuropsychiatric disease, suggesting that SAFB1 is an important regulator of key neuronal processes. The online version of this article (doi:10.1186/s12915-015-0220-7) contains supplementary material, which is available to authorized users.