Department of Pathology and Cell Biology Program, Case Western Reserve University, 2103 Cornell Road, Cleveland, OH 44106, USA.
Department of Pathology and Cell Biology Program, Case Western Reserve University, 2103 Cornell Road, Cleveland, OH 44106, USA.
Curr Biol. 2021 Jun 21;31(12):2507-2519.e4. doi: 10.1016/j.cub.2021.03.041. Epub 2021 Apr 15.
In ribosomal DNA (rDNA) repeats, sequences encoding small-subunit (SSU) rRNA precede those encoding large-subunit (LSU) rRNAs. Processing the composite transcript and subunit assembly requires >100 subunit-specific nucleolar assembly factors (AFs). To investigate the functional organization of the nucleolus, we localized AFs in S. cerevisiae in which the rDNA axis was "linearized" to reduce its dimensionality, thereby revealing its coaxial organization. In this situation, rRNA synthesis and processing continue. The axis is embedded in an inner layer/phase of SSU AFs that is surrounded by an outer layer/phase of LSU AFs. When subunit production is inhibited, subsets of AFs differentially relocate between the inner and outer layers, as expected if there is a cycle of repeated relocation whereby "latent" AFs become "operative" when recruited to nascent subunits. Recognition of AF cycling and localization of segments of rRNA make it possible to infer the existence of assembly intermediates that span between the inner and outer layers and to chart the cotranscriptional assembly of each subunit. AF cycling also can explain how having more than one protein phase in the nucleolus makes possible "vectorial 2-phase partitioning" as a driving force for relocation of nascent rRNPs. Because nucleoplasmic AFs are also present in the outer layer, we propose that critical surface remodeling occurs at this site, thereby partitioning subunit precursors into the nucleoplasm for post-transcriptional maturation. Comparison to observations on higher eukaryotes shows that the coaxial paradigm is likely to be applicable for the many other organisms that have rDNA repeats.
在核糖体 DNA (rDNA) 重复序列中，编码小亚基 (SSU) rRNA 的序列位于编码大亚基 (LSU) rRNA 的序列之前。加工复合转录本和亚基组装需要 >100 种亚基特异性核仁组装因子 (AF)。为了研究核仁的功能组织，我们在 S. cerevisiae 中定位了 AFs，其中 rDNA 轴被“线性化”以降低其维度，从而揭示其同轴组织。在这种情况下，rRNA 合成和加工仍在继续。该轴嵌入在 SSU AF 的内层/相内，该内层/相被 LSU AF 的外层/相包围。当亚基生产受到抑制时，AF 的子集在内外层之间发生差异定位，这是预料之中的，如果存在反复重新定位的循环，那么当被招募到新生亚基时，“潜伏”的 AF 就会变得“活跃”。AF 循环的识别和 rRNA 片段的定位使得推断存在跨越内外层的组装中间体成为可能，并绘制出每个亚基的共转录组装。AF 循环还可以解释为什么核仁中有多个蛋白质相使得新生 rRNP 的重新定位成为可能，这是一种“向量 2 相分区”的驱动力。由于核质 AFs 也存在于外层，我们提出关键的表面重塑发生在这个位置，从而将亚基前体分区到核质中进行转录后成熟。与高等真核生物的观察结果相比，同轴范例可能适用于具有 rDNA 重复序列的许多其他生物体。
