Functional Analysis of the Zinc Finger Modules of the S. cerevisiae Splicing Factor Luc7.
Functional Analysis of the Zinc Finger Modules of the S. cerevisiae Splicing Factor Luc7.
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酿酒酵母剪接因子 Luc7 的锌指模块的功能分析。
DOI：
10.1101/2024.02.04.578419
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Hoskins,AaronA
中科院分区：
文献类型：
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作者：
Carrocci,TuckerJ;DeMario,Samuel;He,Kevin;Zeps,NatalieJ;Harkner,CadeT;Chanfreau,Guillaume;Hoskins,AaronA
剪接位点的鉴定是前信使RNA（pre-mRNA）剪接中的关键步骤，因为外显子/内含子边界的定义控制哪些核苷酸被并入成熟mRNA中。内含子与上游外显子的边界最初通过与U1小核核糖核蛋白（snRNP）的相互作用来识别。这涉及U1 snRNA和前mRNA之间的碱基配对以及snRNP蛋白与5′剪接位点（5′ss）/snRNA双链体的相互作用。在酵母中，这种双链体由两个保守的蛋白因子Yhc 1和Luc 7支持。Luc 7有三个人类旁系同源物（LUC 7 L、LUC 7 L2和LUC 7 L3），它们在选择性剪接中发挥作用。这些旁系同源物的哪些结构域促进特定位点的剪接尚不清楚。在这里，我们人源化的酵母Luc 7蛋白的锌指（ZnF）结构域，以了解其在剪接位点选择中的作用，使用报告分析，转录组分析和遗传相互作用。虽然我们无法确定第一个ZnF结构域的功能，但将第二个ZnF结构域人源化以反映LUC 7 L或LUC 7 L2中发现的功能，导致非共有5′ss的使用发生改变。相反，LUC 7 L3的相应ZnF结构域不能支持酵母活力。此外，Luc 7的人源化可以抑制ATP酶Prp 28的突变，该突变参与U1的释放和在5′ss处与U6的交换。我们的工作揭示了第二个ZnF的Luc 7剪接位点选择的作用，并表明不同的ZnF域可能有不同的ATP酶的要求释放Prp 28。
Identification of splice sites is a critical step in pre-messenger RNA (pre-mRNA) splicing because the definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 small nuclear ribonucleoprotein (snRNP). This involves both base-pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5′ splice site (5′ss)/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3), which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger (ZnF) domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. Although we were unable to determine a function for the first ZnF domain, humanization of the second ZnF domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5′ss. In contrast, the corresponding ZnF domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5′ss. Our work reveals a role for the second ZnF of Luc7 in splice site selection and suggests that different ZnF domains may have different ATPase requirements for release by Prp28.