Centre for Genetics and Genomics, School of Biology, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK.
Chromosome Res. 2010 Dec;18(8):851-72. doi: 10.1007/s10577-010-9170-7. Epub 2010 Nov 18.
RNA-binding motif (RBM) proteins comprise a large family of RNA-binding proteins whose functions are poorly understood. Since some RBM proteins are candidate alternative splicing factors we examined whether one such member of the family, RBM6, exhibited a pattern of nuclear distribution and targeting consistent with this role. Using antibodies raised against mouse RBM6 to immunostain mammalian cell lines we found that the endogenous protein was both distributed diffusely in the nucleus and concentrated in a small number of nuclear foci that corresponded to splicing speckles/interchromatin granule clusters (IGCs). Tagged RBM6 was also targeted to IGCs, although it accumulated in large bodies confined to the IGC periphery. The basis of this distribution pattern was suggested by the targeting of tagged RBM6 in the giant nuclei (or germinal vesicles (GVs)) of Xenopus oocytes. In spread preparations of GV contents RBM6 was localized both to lampbrush chromosomes and to the surface of many oocyte IGCs, where it was confined to up to 50 discrete patches. Each patch of RBM6 labelling corresponded to a bead-like structure of 0.5-1 microm diameter that assembled de novo on the IGC surface. Assembly of these novel structures depended on the repetitive N-terminal region of RBM6, which acts as a multimerization domain. Without this domain, RBM6 was no longer excluded from the IGC interior but accumulated homogeneously within it. Assembly of IGC-surface structures in mammalian cell lines also depended on the oligomerization domain of RBM6. Oligomerization of RBM6 also had morphological effects on its other major target in GVs, namely the arrays of nascent transcripts visible in lampbrush chromosome transcription units. The presence of oligomerized RBM6 on many lampbrush loops caused them to appear as dense structures with a spiral morphology that appeared quite unlike normal, extended loops. This distribution pattern suggests a new role for RBM6 in the co-transcriptional packaging or processing of most nascent transcripts.
RNA 结合基序 (RBM) 蛋白是一大类 RNA 结合蛋白家族，其功能尚未完全了解。由于某些 RBM 蛋白是候选可变剪接因子，我们研究了家族中的一个成员，RBM6，是否表现出与其作用一致的核分布和靶向模式。使用针对小鼠 RBM6 制备的抗体对哺乳动物细胞系进行免疫染色，我们发现内源性蛋白在核内弥散分布，并集中在少数核斑点中，这些核斑点与剪接斑点/核间颗粒簇（IGC）相对应。标记的 RBM6 也被靶向到 IGC，但它在局限于 IGC 外周的大体内积累。这种分布模式的基础是通过在非洲爪蟾卵母细胞的巨核（或生发泡 (GV)）中靶向标记的 RBM6 来提示的。在 GV 内容物的展开制剂中，RBM6 定位于灯刷染色体和许多卵母细胞 IGC 的表面，在那里它局限于多达 50 个离散的斑点。每个 RBM6 标记的斑点对应于一个直径为 0.5-1 微米的珠状结构，该结构在 IGC 表面从头组装。这些新结构的组装取决于 RBM6 的重复 N 端区域，该区域充当多聚化结构域。没有这个结构域，RBM6 不再被排除在 IGC 内部，但均匀地积累在内部。哺乳动物细胞系中 IGC 表面结构的组装也依赖于 RBM6 的寡聚化结构域。RBM6 的寡聚化对其在 GV 中的另一个主要靶标，即在灯刷染色体转录单位中可见的新生转录物的阵列，也具有形态学影响。许多灯刷环上聚集的 RBM6 使其看起来像密集结构，具有螺旋形态，与正常的延伸环完全不同。这种分布模式表明 RBM6 在大多数新生转录物的共转录包装或加工中具有新的作用。
