McMurdie Karen, Peeney Allison N, Mefford Melissa A, Baumann Peter, Zappulla David C
Department of Biology, Johns Hopkins University, Baltimore, Maryland, USA.
Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania, USA.
bioRxiv. 2025 Feb 23:2025.02.22.638514. doi: 10.1101/2025.02.22.638514.
The telomerase RNA-protein enzyme is critical for most eukaryotes to complete genome copying by extending chromosome ends, thus solving the end-replication problem and postponing senescence. Despite the importance of the fission yeast to biomedical research, very little is known about the structure of its 1212 nt telomerase RNA. We have determined the secondary structure of this large RNA, TER1, based on phylogenetics and bioinformatic modeling, as well as genetic and biochemical analyses. We find that several conserved regions of the rapidly evolving TER1 RNA are important for the ability of telomerase to maintain telomeres, based on testing truncation mutants , whereas, overall, many other large regions are dispensable. This is similar to budding yeast telomerase RNA, TLC1, and consistent with functioning as a flexible scaffold for the RNP. We tested if the essential three-way junction works from other locations in TER1, finding that indeed it can, supporting that it is flexibly scaffolded. Furthermore, we find that a half-sized Mini-TER1 allele, built from the catalytic core and the three-way junction, reconstitutes catalytic activity with TERT . Overall, we provide a secondary structure model for the large fission-yeast telomerase lncRNA based on phylogenetics and molecular-genetic testing in cells and insight into the RNP's physical and functional organization.
端粒酶RNA - 蛋白质酶对于大多数真核生物通过延长染色体末端来完成基因组复制至关重要，从而解决了末端复制问题并延缓了衰老。尽管裂殖酵母对生物医学研究很重要，但对其1212个核苷酸的端粒酶RNA的结构却知之甚少。我们基于系统发育学、生物信息学建模以及遗传和生化分析，确定了这种大RNA即TER1的二级结构。基于对截短突变体的测试，我们发现快速进化的TER1 RNA的几个保守区域对于端粒酶维持端粒的能力很重要，而总体而言，许多其他大区域是可有可无的。这与芽殖酵母端粒酶RNA即TLC1类似，并且与作为RNP的灵活支架发挥功能一致。我们测试了TER1中其他位置的必需三向接头是否起作用，发现它确实可以，这支持了它是灵活构建的。此外，我们发现由催化核心和三向接头构建的半尺寸Mini - TER1等位基因与TERT一起重建了催化活性。总体而言，我们基于系统发育学以及细胞中的分子遗传学测试，为大的裂殖酵母端粒酶lncRNA提供了一个二级结构模型，并深入了解了RNP的物理和功能组织。
