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.

中文翻译：

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拟南芥拥有两个新的 ELL 相关因子同源物

转录延伸是在真核细胞中调节 RNA 聚合酶 II 指导的蛋白质编码基因表达的关键步骤之一。不同的蛋白质已被证明可以控制这一过程，包括 ELL/EAF 家族。ELL 相关因子 (EAFs) 首次在酵母双杂交筛选中发现，作为人类 ELL（11 种富含赖氨酸的白血病）转录延伸因子的相互作用伙伴。随后，它们在不同的生物体中被发现，包括粟酒裂殖酵母。然而，尚未从植物中表征 EAF 的同源物。在目前的工作中，我们在不同植物中鉴定了 EAF 直系同源序列，并表征了两种新的拟南芥EAF 同源物，AtEAF-1 (At1g71080) 和 AtEAF-2 (At5g38050)。序列分析表明，AtEAF-1 和 AtEAF-2 都与其对应的S. pombe EAF 表现出相似性。此外，拟南芥和庞贝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。我们证明 AtEAF-2，但不是 AtEAF-1 在功能上补充了粟酒裂殖酵母 eaf突变体的生长缺陷。我们还表明，只有 AtEAF-1 显示出类似于S. pombe EAF 直向同源物的反式激活潜力。随后在拟南芥中的表达分析表明这两种同源物在不同发育阶段和研究中测试的不同组织中以不同水平表达。AtEAF-1或AtEAF-2的单个无效突变体在发育上是正常的，这意味着它们的功能冗余。总之，我们的结果提供了第一个证据表明拟南芥也具有功能性 EAF 蛋白，这表明这些蛋白质在生物体中具有进化保守性。