RNA. 2013 Oct;19(10):1419-31. doi: 10.1261/rna.038547.113. Epub 2013 Aug 23.
Although biogenesis of ribosomes is a crucial process in all organisms and is thus well conserved, Trypanosoma brucei ribosome biogenesis, of which maturation of rRNAs is an early step, has multiple points of divergence. Our aim was to determine whether in the processing of the pre-rRNA precursor molecule, 5'→3' exoribonuclease activity in addition to endonucleolytic cleavage is necessary in T. brucei as in other organisms. Our approach initiated with the bioinformatic identification of a putative 5'→3' exoribonuclease, XRNE, which is highly diverged from the XRN2/Rat1 enzyme responsible for rRNA processing in other organisms. Tagging this protein in vivo allowed us to classify XRNE as nucleolar by indirect immunofluorescence and identify by copurification interacting proteins, many of which were ribosomal proteins, ribosome biogenesis proteins, and/or RNA processing proteins. To determine whether XRNE plays a role in ribosome biogenesis in procyclic form cells, we inducibly depleted the protein by RNA interference. This resulted in the generation of aberrant preprocessed 18S rRNA and 5' extended 5.8S rRNA, implicating XRNE in rRNA processing. Polysome profiles of XRNE-depleted cells demonstrated abnormal features including an increase in ribosome small subunit abundance, a decrease in large subunit abundance, and defects in polysome assembly. Furthermore, the 5' extended 5.8S rRNA in XRNE-depleted cells was observed in the large subunit, monosomes, and polysomes in this gradient. Therefore, the function of XRNE in rRNA processing, presumably due to exonucleolytic activity very early in ribosome biogenesis, has consequences that persist throughout all biogenesis stages.
尽管核糖体的生物发生是所有生物体中的一个关键过程，因此得到了很好的保守，但锥虫核糖体的生物发生，其中 rRNA 的成熟是早期步骤，具有多个分歧点。我们的目的是确定在 pre-rRNA 前体分子的加工过程中，是否除了内切核酸酶切割之外，5'→3'外切核酸酶活性在 T. brucei 中是必需的，就像在其他生物体中一样。我们的方法首先从生物信息学上鉴定了一个假定的 5'→3'外切核酸酶 XRNE，它与负责其他生物体中 rRNA 加工的 XRN2/Rat1 酶高度分化。在体内标记这种蛋白质，使我们能够通过间接免疫荧光将 XRNE 归类为核仁，并通过共纯化鉴定相互作用的蛋白质，其中许多是核糖体蛋白、核糖体生物发生蛋白和/或 RNA 加工蛋白。为了确定 XRNE 是否在裂殖体形式的细胞中核糖体生物发生中发挥作用，我们通过 RNA 干扰诱导性地耗尽该蛋白质。这导致异常的预处理 18S rRNA 和 5'延伸的 5.8S rRNA 的产生，表明 XRNE 参与 rRNA 加工。XRNE 耗尽细胞的多核糖体图谱显示出异常特征，包括核糖体小亚基丰度增加、大亚基丰度降低以及多核糖体组装缺陷。此外，在 XRNE 耗尽的细胞中，5'延伸的 5.8S rRNA 也观察到大亚基、单核糖体和多核糖体在这个梯度中。因此，XRNE 在 rRNA 加工中的功能，可能由于核糖体生物发生中非常早期的外切核酸酶活性，在整个生物发生阶段都有影响。
