Saturation mutagenesis reveals manifold determinants of exon definition.
Saturation mutagenesis reveals manifold determinants of exon definition.
复制标题
DOI：
10.1101/gr.219683.116
复制
发表时间：
2018-01
期刊：
影响因子：
7
通讯作者：
Chasin LA
中科院分区：
文献类型：
--
作者：
Ke S;Anquetil V;Zamalloa JR;Maity A;Yang A;Arias MA;Kalachikov S;Russo JJ;Ju J;Chasin LA
为了阐明外显子序列在人类 RNA 转录本剪接中的程度和作用，我们对三外显子小基因中的 51 nt 内外显子进行了饱和诱变。对所有可能的单核苷酸和串联二核苷酸取代进行了调查。使用高通量遗传学，对 5560 个小基因分子在人 HEK293 细胞中的剪接进行了分析。高达 70% 的突变产生大量（超过两倍）剪接增加或减少的表型。在所有预测的二级结构元件中，只有一个 15-nt 茎环显示出与剪接的强相关性，表现出负相关。突变分子和与剪接体形成相关的蛋白质（U2AF35、U2AF65、U1A 和 U1-70K）之间的外显子-蛋白质复合物的体外形成与剪接效率相关，表明外显子定义是受大多数突变影响的步骤。 CISBP-RNA 数据库中报告的数十个人类 RNA 结合蛋白结构域的测量相对结合亲和力被发现与剪接效率呈正相关或负相关，超过了同时适合 51 nt 测试外显子的程度。大量的功能蛋白结合相关性表明前体 mRNA 分子是动态且异质的群体，每个分子都对特定的结合蛋白集合做出反应。
To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem–loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins.
登录
查看更多内容
影响因子：
9.8
作者：
Fairbrother WG;Holste D;Burge CB;Sharp PA
通讯作者：
Sharp PA
DOI：
10.1073/pnas.1017700108
发表时间：
2011-05-17
影响因子：
11.1
作者：
Cho, Suhyung;Hoang, Amy;Ghosh, Gourisankar
通讯作者：
Ghosh, Gourisankar
影响因子：
7
作者：
Ke, Shengdong;Shang, Shulian;Chasin, Lawrence A.
通讯作者：
Chasin, Lawrence A.
影响因子：
4.5
作者：
Cavaloc, Y;Bourgeois, CF;Stévenin, J
通讯作者：
Stévenin, J
DOI：
10.1186/1748-7188-6-26
发表时间：
2011-11-24
期刊：
Algorithms for molecular biology : AMB
影响因子：
--
作者：
Lorenz R;Bernhart SH;Höner Zu Siederdissen C;Tafer H;Flamm C;Stadler PF;Hofacker IL
通讯作者：
Hofacker IL