Spatial organisation of nuclear compartments is an important regulator of chromatin function, yet the molecular principles that maintain nuclear architecture remain ill-defined. We have used RNA interference to deplete key structural nuclear proteins, the nuclear lamins. In HeLa cells, we show that reduced expression of lamin B1, but not lamin A/C, severely inhibits RNA synthesis--first by RNA polymerase II and later by RNA polymerase I. Declining levels of transcription correlate with different morphological changes in major nuclear compartments, nucleoli and nuclear speckles. Ultimately, nuclear changes linked to the loss of synthetic activity result in expansion of the inter-chromatin domain and corresponding changes in the structure and spatial organisation of chromosome territories, which relocate towards the nuclear periphery. These results show that a lamin B1-containing nucleoskeleton is required to maintain RNA synthesis and that ongoing synthesis is a fundamental determinant of global nuclear architecture in mammalian cells.