Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.
Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.
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DOI：
10.1371/journal.pone.0242725
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发表时间：
2020
期刊：
影响因子：
3.7
通讯作者：
Braddock DT
中科院分区：
文献类型：
--
作者：
Hsiao HT;Crichlow GV;Murphy JW;Folta-Stogniew EJ;Lolis EJ;Braddock DT
mRNA 前体剪接对于在给定时间获得所需量的转录物以及调节编码蛋白的产生至关重要。给定的前 mRNA 可以通过多种方式进行剪接，或者根本不进行剪接，从而产生多种基因产物。许多剪接因子被招募到前 mRNA 剪接位点以确保正确剪接。其中一种因子，60 kDa 聚（U）结合剪接因子（PUF60），被招募到并不总是剪接的位点，而是充当替代剪接位点。在本研究中，我们表征了 PUF60 与腺病毒主要晚期启动子剪接位点（AdML 3' 剪接位点，AdML3'）的相互作用。我们发现 PUF60-AdML3 的解离常数在微摩尔范围内，结合亲和力主要由 PUF60 的两个中心 RNA 识别基序 (RRM) 提供。与 AdML3 复合的两个 PUF60 RRM 的 1.95 Å 晶体结构揭示了一种二聚体组织，将两段核酸束置于相反的方向，这可能导致核酸成环，并解释了 PUF60 如何影响前 mRNA 几何形状以实现剪接。通过光散射和紫外/可见光谱对该复合物的溶液表征表明潜在的 2:1 (PUF602:AdML3') 化学计量，与晶体结构一致。这项工作定义了前 mRNA 3' 剪接位点的选择性剪接因子 PUF60 的序列特异性。我们的观察表明，对前 mRNA 方向性的控制在剪接体组装的早期阶段非常重要，并增进了我们对选择性剪接因子和组成型剪接因子在 3' 剪接位点之间区分的分子机制的理解。
Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3’). We found that the PUF60–AdML3’ dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60’s two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3’ revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3’) stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3’ splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3’ splice sites.
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影响因子：
11.4
作者：
Crichlow, Gregg V.;Zhou, Hongwen;Hsiao, Hsin-hao;Frederick, Kendra B.;Debrosse, Maxime;Yang, Yuande;Folta-Stogniew, Ewa J.;Chung, Hye-Jung;Fan, Chengpeng;De La Cruz, Enrique M.;Levens, David;Lolis, Elias;Braddock, Demetrios
通讯作者：
Braddock, Demetrios
影响因子：
5.3
作者：
Guth, S;Tange, TO;Valcárcel, J
通讯作者：
Valcárcel, J
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL
影响因子：
4.8
作者：
Kent, OA;Reayi, A;MacMillan, AM
通讯作者：
MacMillan, AM
DOI：
10.1038/nrg2673
发表时间：
2010-01
期刊：
Nature reviews. Genetics
影响因子：
--
作者：
通讯作者：
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