RCC1 and nuclear organization.

Abstract

We have examined the effect of RCC1 function on the nuclear organization of pre-mRNA splicing factors and poly(A)+ RNA in the tsBN2 cells, a RCC1 temperature-sensitive mutant cell line. We have found that at 4-6 h after shifting cells from the permissive temperature (32.5°C) to the restrictive temperature (39.5°C), both small nuclear ribonucleoprotein particles and a general splicing factor SC35 reorganized into 4-10 large round clusters in the nucleus, as compared with the typical speckled distribution seen in cells at the permissive temperature. In situ hybridization to poly(A)+ RNA resulted in a similar pattern. Examination by double labeling demonstrated that the redistribution of splicing factors coincides with that of poly(A)+ RNA. Such changes in the nuclear organization of splicing factors and poly(A)+ RNA were not the result of the temperature shift or of chromatin condensation. Cellular transcription was not significantly altered in these cells and extracts made from both the permissive and restrictive temperature were splicing competent. Electron microscopic examination demonstrated that the large clusters containing both splicing factors and poly(A)+ RNA were fused interchromatin granule clusters. In addition, small electron-dense dot-like structures measuring approximately 80 nm in diameter were also observed, most of which are accumulated in enlarged interchromatin granule clusters in the nucleoplasm of RCC1 -cells. In spite of the significant changes observed in the nucleoplasm, relatively little alteration was observed in nucleolar structure by both light and electron microscopic examination. The above observations suggest that the RCC1 protein directly or indirectly regulates the organization of splicing components and poly(A)+ RNA in the cell nucleus and that RCC1 may play a role in nuclear organization.

Nuclear RanGTP is not required for targeting small nucleolar RNAs to the nucleolus

“…To verify that the cells have undergone the temperature shift, the localization of the trimethyl guanosine cap (TMG), a marker for the TMG-capped U snRNAs, was also examined. As previously reported, U snRNA localization shifts from widespread nucleoplasmic staining to less numerous foci when the RanGTP gradient is disrupted (Cheng et al, 1995;Huang et al, 1997). These data suggest that in contrast to previous reports (Cheng et al, 1995), the RanGTP gradient may not be required for localization of snoRNAs to the nucleolus.…”

“…Previous work has shown that the temperature-sensitive RCC1 mutant, tsBN2, shows a change in the intra-nuclear localization of both small ribonucleoproteins (snRNPs) and the general splicing factor SC35 (Cheng et al, 1995;Huang et al, 1997) following a shift to the non-permissive temperature. Furthermore, biochemical experiments have implicated RCC1 in the transport of some but not all RNAs within the nucleus (Cheng et al, 1995).…”

ETO-2 Associates with SCL in Erythroid Cells and Megakaryocytes and Provides Repressor Functions in Erythropoiesis

“…3B, merge) and L8057 (data not shown) cells. As controls, we also show colocalization of SCL and known partner Ldb-1, but not of an abundant unrelated nuclear protein (splicing factor SC35 [32]) with a component of the SCL complex such as Ldb-1 (Fig. 3B).…”

Evidence that Myb-related CDC5 proteins are required for pre-mRNA splicing

“…29) should similarly modulate the subnuclear localization of mammalian CDC5. In particular, tsBN2 cells (12) reorganize splicing factors into 4-10 large clusters at the restrictive temperature (13). To determine whether the CDC5 distribution is similarly reorganized, tsBN2 cells were shifted to 39.5°C for 10 h and costained for SC35 and CDC5.…”

“…TsBN2 cells (12) were cultured and shifted to the restrictive temperature for 10 h as described (13). Asynchronously growing subconfluent NIH 3T3 cells were rendered quiescent by incubation for 24 h in DMEM containing 0.1% bovine calf serum.…”