A shared RNA-binding site in the Pet54 protein is required for translational activation and group I intron splicing in yeast mitochondria.
A shared RNA-binding site in the Pet54 protein is required for translational activation and group I intron splicing in yeast mitochondria.
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DOI：
10.1093/nar/gkn045
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发表时间：
2008-05
影响因子：
14.9
通讯作者：
Caprara MG
中科院分区：
文献类型：
--
作者：
Kaspar BJ;Bifano AL;Caprara MG
Pet54p蛋白是双重功能（“兼职”）蛋白的典型例子：在酿酒酵母线粒体（mt）中，它是COX3 mRNA翻译激活和COX1前mRNA中aI5β组I内含子剪接所必需的。酵母的遗传和生化分析与Pet54p与其他翻译激活因子形成复合物一致，该复合物以未知的方式与COX3 mRNA的5 '未翻译先导体（UTL）相关。同样，遗传分析表明，Pet54p与另一组不同的蛋白质一起促进aI5β内含子的剪接，但Pet54的功能也不清楚。特别是，目前尚不清楚Pet54p是否是特异性识别5 ' UTL和内含子rna的主要rna结合蛋白，或者其功能特异性是否以其他方式控制。利用重组蛋白，我们发现Pet54p与aI5β内含子结合具有高特异性和亲和力，并促进体外外显子连接。此外，Pet54p与COX3 5 ' UTL RNA结合具有相似的亲和力。竞争实验表明，COX3 5'UTL和aI5β内含子rna结合在Pet54p上相同或重叠的表面。通过RNA缺失和RNase足迹对Pet54p结合位点的描述表明，Pet54p结合在两种RNA中具有相似的长度序列。序列比对显示，结合位点之间存在显著的相似性和重叠（56%）。鉴于其在剪接中的作用可能是一种获得性功能，这些数据支持Pet54p的剪接功能可能是由于与aI5β内含子的偶然结合而产生的模型。这种关联可能导致了Pet54p变异的选择，从而提高了aI5β剪接的效率，并为COX1和COX3的表达提供了一种可能的途径。
The Pet54p protein is an archetypical example of a dual functioning (‘moonlighting’) protein: it is required for translational activation of the COX3 mRNA and splicing of the aI5β group I intron in the COX1 pre-mRNA in Saccharomyces cerevisiae mitochondria (mt). Genetic and biochemical analyses in yeast are consistent with Pet54p forming a complex with other translational activators that, in an unknown way, associates with the 5′ untranslated leader (UTL) of COX3 mRNA. Likewise, genetic analysis suggests that Pet54p along with another distinct set of proteins facilitate splicing of the aI5β intron, but the function of Pet54 is, also, obscure. In particular, it remains unknown whether Pet54p is a primary RNA-binding protein that specifically recognizes the 5′ UTL and intron RNAs or whether its functional specificity is governed in other ways. Using recombinant protein, we show that Pet54p binds with high specificity and affinity to the aI5β intron and facilitates exon ligation in vitro. In addition, Pet54p binds with similar affinity to the COX3 5′ UTL RNA. Competition experiments show that the COX3 5′UTL and aI5β intron RNAs bind to the same or overlapping surface on Pet54p. Delineation of the Pet54p-binding sites by RNA deletions and RNase footprinting show that Pet54p binds across a similar length sequence in both RNAs. Alignment of the sequences shows significant (56%) similarity and overlap between the binding sites. Given that its role in splicing is likely an acquired function, these data support a model in which Pet54p's splicing function may have resulted from a fortuitous association with the aI5β intron. This association may have lead to the selection of Pet54p variants that increased the efficiency of aI5β splicing and provided a possible means to coregulate COX1 and COX3 expression.
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影响因子：
2.9
作者：
Emerick, VL;Pan, J;Woodson, SA
通讯作者：
Woodson, SA
影响因子：
2.9
作者：
Golden, BL;Cech, TR
通讯作者：
Cech, TR
DOI：
10.1007/bf01952112
发表时间：
1996-12-01
期刊：
EXPERIENTIA
影响因子：
--
作者：
Fox, TD
通讯作者：
Fox, TD
影响因子：
2.9
作者：
RYAN, PC;DRAPER, DE
通讯作者：
DRAPER, DE
影响因子：
3.9
作者：
Sanford, JR;Ellis, J;C치ceres, JF
通讯作者：
C치ceres, JF