Carrocci Tucker J, DeMario Samuel, He Kevin, Zeps Natalie J, Harkner Cade T, Chanfreau Guillaume, Hoskins Aaron A
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
bioRxiv. 2024 Feb 4:2024.02.04.578419. doi: 10.1101/2024.02.04.578419.
Identification of splice sites is a critical step in pre-mRNA splicing since 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 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/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 domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. While we were unable to determine a function for the first zinc finger domain, humanization of the second zinc finger domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5' splice sites. In contrast, the corresponding zinc finger 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' splice site. Our work reveals a role for the second zinc finger of Luc7 in splice site selection and suggests that different zinc finger domains may have different ATPase requirements for release by Prp28.
剪接位点的识别是前体mRNA剪接中的关键步骤，因为外显子/内含子边界的界定决定了哪些核苷酸会被纳入成熟mRNA中。内含子与上游外显子的边界最初是通过与U1 snRNP的相互作用来识别的。这涉及U1 snRNA与前体mRNA之间的碱基配对，以及snRNP蛋白与5'剪接位点/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质因子Yhc1和Luc7支撑。Luc7有三个人类同源物（LUC7L、LUC7L2和LUC7L3），它们在可变剪接中发挥作用。这些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，我们对酵母Luc7蛋白的锌指结构域进行了人源化，以便使用报告基因检测、转录组分析和遗传相互作用来了解它们在剪接位点选择中的作用。虽然我们无法确定第一个锌指结构域的功能，但将第二个锌指结构域人源化为与LUC7L或LUC7L2中发现的结构域相同，导致了非共识5'剪接位点使用的改变。相比之下，LUC7L3的相应锌指结构域不能支持酵母的生存能力。此外，Luc7的人源化可以抑制ATP酶Prp28的突变，Prp28参与U1在5'剪接位点的释放和与U6的交换。我们的工作揭示了Luc7的第二个锌指在剪接位点选择中的作用，并表明不同的锌指结构域可能对Prp28释放有不同的ATP酶需求。
