Prenucleolar bodies contain coilin and are assembled in Xenopus egg extract depleted of specific nucleolar proteins and U3 RNA

Abstract

Nuclei assembled in Xenopus egg extract contain numerous spherical aggregations or nuclear bodies. Previously we have shown that they closely resemble prenucleolar bodies (PNBs), both at the compositional and ultrastructural level. Subsequently, coilin was also identified and for this reason they were called coiled bodies. Here we present morphological and immunocytochemical evidence that the in vitro nuclear bodies resemble authentic PNBs and are different from coiled bodies. In particular we show that coilin, previously considered as the defining protein constituent of coiled bodies, is also present in PNBs of cultured cells. In contrast, the PNB-associated nucleolar proteins nucleolin and B23/NO38 are not detectable in coiled bodies and may thus serve as suitable markers for PNBs. Our results suggest that PNBs are primary assembly structures which contribute to the formation of both nucleoli and coiled bodies and thus offer an explanation for the frequently observed structural association of coiled bodies with nucleoli. To gain some insight into the assembly process of PNBs in vitro, specific nucleolar proteins were removed from Xenopus egg extract. Quite surprisingly, the immuno-depleted extracts still promoted the assembly of nuclear bodies which lacked either fibrillarin, nucleolin, xNopp180 or B23/NO38. Only after fibrillarin-depletion fewer PNBs were seen as compared to controls. Digestion of the extract with RNase followed by northern blot analysis revealed that U3 small nucleolar RNA is not required for the formation and structural maintenance of PNBs in vitro.