Dynamics of WD-repeat containing proteins in SSU processome components.

Nine WD-repeat containing proteins in human SSU processome components have been found in a HeLa cell nuclear matrix fraction. In these proteins, t-UTP sub-complex components, i.e., CIRH1A, UTP15, and WDR43, were shown to be immobilized in the fibrillar centers of nucleoli in living cells. In this study, the dynamics of the remaining six proteins fused with green fluorescent protein (GFP), i.e., PWP2-GFP, TBL3-GFP, GFP-UTP18, GFP-NOL10, GFP-WDR46, and GFP-WDSOF1, were examined in living cells. The findings were as follows. (i) The majority of UTP-B sub-complex components, i.e., PWP2-GFP, TBL3-GFP, and GFP-UTP18, are localized to the dense fibrillar component and granular component regions in nucleoli; (ii) When rRNA transcription is suppressed, the majority of GFP-fused UTP-B sub-complex components are localized in the cap and body regions of nucleoli. (iii) The mobility of these proteins except for GFP-WDSOF1, and half of GFP-UTP18 and GFP-WDR46, respectively, is very low in living cells. (iv) When rRNA transcription is suppressed, the mobility of these proteins except for GFP-WDSOF1 is accelerated but still slow. These findings and others suggest that these WD-repeat proteins other than GFP-WDSOF1 found in the nuclear matrix fraction bind tightly to some macro-protein complexes and act as a scaffold or a core for the complexes in nucleoli.

Interaction, mobility, and phosphorylation of human orthologues of WD repeat-containing components of the yeast SSU processome t-UTP sub-complex.

We previously proposed a dynamic scaffold model for inner nuclear structure formation. In this model, structures in inter-chromatin regions are maintained through dynamic interaction of protein complex modules, and WD repeat- and disordered region-rich proteins and others act as scaffolds for these protein complexes. In this study, three WD-repeat proteins, i.e., CIRH1A, UTP15, and WDR43, were found in the nuclear matrix fraction and speculated to be present in the human t-UTP sub-complex of SSU processomes. The results obtained as to their subnuclear localization, binding with each other, mobilities, and phosphorylation were: (i) the majority of these proteins fused with GFP are localized to the fibrillar center region in nucleoli. (ii) these 3 proteins bind directly with each other in vitro. (iii) the movement of these proteins is very slow in living cells and independent of rDNA transcription. (iv) His-CIRH1A is phosphorylated at Thr(131) by a mitotic Xenopus egg extract, and binding with GST-UTP15 and GST-WDR43 is suppressed. These findings and others suggest that these 3 WD proteins found in the matrix fraction bind directly with each other, bind tightly to fibrillar center regions, and comprise a part of the nucleolar structure. These results are also consistent with our dynamic scaffold model.