RCC1 AND NUCLEAR-ORGANIZATION

We have examined the effect of RCC1 function on the nuclear organizati on 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 degre es C) to the restrictive temperature (39.5 degrees C), both small nucl ear ribonucleoprotein particles and a general splicing factor SC35 reo rganized into 4-10 large round clusters in the nucleus, as compared wi th the typical speckled distribution seen in cells at the permissive t emperature. In situ hybridization to poly(A)(+) RNA resulted in a simi lar pattern. Examination by double labeling demonstrated that the redi stribution 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 restrictiv e temperature were splicing competent. Electron microscopic examinatio n demonstrated that the large clusters containing both splicing factor s and poly(A)(+) RNA were fused interchromatin granule clusters. In ad dition, small electron-dense dot-like structures measuring approximate ly 80 nm in diameter were also observed, most of which are accumulated in enlarged interchromatin granule clusters in the nucleoplasm of RCC 1- cells. In spite of the significant changes observed in the nucleopl asm, relatively little alteration was observed in nucleolar structure by both light and electron microscopic examination. The above observat ions suggest that the RCC1 protein directly or indirectly regulates th e organization of splicing components and poly(A)(+) RNA in the cell n ucleus and that RCC1 may play a role in nuclear organization.