University School of Biotechnology, G.G.S. Indraprastha University, New Delhi, India.
Institute of Life Sciences, Bhubaneshwar, Odisa, India.
IUBMB Life. 2021 Sep;73(9):1115-1130. doi: 10.1002/iub.2513. Epub 2021 Jun 15.
Transcription elongation is one of the key steps at which RNA polymerase II-directed expression of protein-coding genes is regulated in eukaryotic cells. Different proteins have been shown to control this process, including the ELL/EAF family. ELL Associated Factors (EAFs) were first discovered in a yeast two-hybrid screen as interaction partners of the human ELL (Eleven nineteen Lysine-rich Leukemia) transcription elongation factor. Subsequently, they have been identified in different organisms, including Schizosaccharomyces pombe. However, no homolog(s) of EAF has as yet been characterized from plants. In the present work, we identified EAF orthologous sequences in different plants and have characterized two novel Arabidopsis thaliana EAF homologs, AtEAF-1 (At1g71080) and AtEAF-2 (At5g38050). Sequence analysis showed that both AtEAF-1 and AtEAF-2 exhibit similarity with its S. pombe EAF counterpart. Moreover, both Arabidopsis thaliana and S. pombe EAF orthologs share conserved sequence characteristic features. Computational tools also predicted a high degree of disorder in regions towards the carboxyl terminus of these EAF proteins. We demonstrate that AtEAF-2, but not AtEAF-1 functionally complements growth deficiencies of Schizosaccharomyces pombe eaf mutant. We also show that only AtEAF-1 displays transactivation potential resembling the S. pombe EAF ortholog. Subsequent expression analysis in A. thaliana showed that both homologs were expressed at varying levels during different developmental stages and in different tissues tested in the study. Individual null-mutants of either AtEAF-1 or AtEAF-2 are developmentally normal implying their functional redundancy. Taken together, our results provide first evidence that A. thaliana also possesses functional EAF proteins, suggesting an evolutionary conservation of these proteins across organisms.
转录延伸是真核细胞中 RNA 聚合酶 II 指导的蛋白质编码基因表达调控的关键步骤之一。已经证明不同的蛋白质可以控制这个过程,包括 ELL/EAF 家族。ELL 相关因子(EAFs)最初是在酵母双杂交筛选中作为人类 ELL(Eleven nineteen Lysine-rich Leukemia)转录延伸因子的相互作用伙伴而被发现的。随后,它们在不同的生物体中被鉴定出来,包括酿酒酵母。然而,目前还没有从植物中鉴定出 EAF 的同源物。在本工作中,我们在不同的植物中鉴定了 EAF 直系同源序列,并对两个新的拟南芥 EAF 同源物 AtEAF-1(At1g71080)和 AtEAF-2(At5g38050)进行了特征描述。序列分析表明,AtEAF-1 和 AtEAF-2 与它们的酿酒酵母 EAF 对应物具有相似性。此外,拟南芥和酿酒酵母 EAF 直系同源物都具有保守的序列特征。计算工具还预测了这些 EAF 蛋白羧基末端区域的高度无序性。我们证明,AtEAF-2 但不是 AtEAF-1 能够功能性地弥补酿酒酵母 eaf 突变体的生长缺陷。我们还表明,只有 AtEAF-1 显示出类似于酿酒酵母 EAF 同源物的转录激活潜力。随后在拟南芥中的表达分析表明,在本研究中测试的不同发育阶段和不同组织中,两个同源物的表达水平都有所不同。AtEAF-1 或 AtEAF-2 的单个缺失突变体在发育上是正常的,这表明它们的功能冗余。综上所述,我们的结果首次提供了证据表明拟南芥也拥有功能性的 EAF 蛋白,表明这些蛋白在不同的生物体中具有进化上的保守性。