Mutational analysis of p80 coilin indicates a functional interaction between coiled bodies and the nucleolus.

Abstract

Abstract. Coiled bodies are conserved subnuclear domains found in both plant and animal cells. They contain a subset of splicing snRNPs and several nucleolar antigens, including Nopp140 and fibrillarin. In addition, autoimmune patient sera have identified a coiled body specific protein, called p80 coilin. In this study we show that p80 coilin is ubiquitously expressed in human tissues. The full-length human p80 coilin protein correctly localizes in coiled bodies when exogenously expressed in HeLa cells using a transient transfection assay. Mutational analysis identifies separate domains in the p80 coilin protein that differentially affect its subnuclear localization. The data show that p80 coilin has a nuclear localization signal, but this is not sufficient to target the protein to coiled bodies. The results indicate that localization in coiled bodies is not determined by a simple motif analogous to the NLS motifs involved in nuclear import. A specific carboxy-terminal deletion in p80 coilin results in the formation of pseudo-coiled bodies that are unable to recruit splicing snRNPs. This causes a loss of endogenous coiled bodies. A separate class of mutant coilin proteins are shown to localize in fibrillar structures that surround nucleoli. These mutants also lead to loss of endogenous coiled bodies, produce a dramatic disruption of nucleolar architecture and cause a specific segregation of nucleolar antigens. The structural change in nucleoli is accompanied by the loss of RNA polymerase I activity. These data indicate that p80 coilin plays an important role in subnuclear organization and suggest that there may be a functional interaction between coiled bodies and nucleoli.T HE nucleus is a highly compartmentalized organelle.There is now evidence that protein and RNP factors involved in nuclear processes such as transcription, replication, pre-mRNA processing, and ribosome biogenesis are either partially or exclusively localized within discrete subnuclear domains or compartments (for review see Spector, 1993). In addition, several subnuclear compartments are known whose functions have not yet been identified. These include, inter alia, snRNP containing structures, such as coiled bodies and interchromatin granule clusters (for reviews see Gall, 1991;Brasch and Ochs, 1992;Lamond and Carmo-Fonseca, 1993;Spector, 1993;Bohmann et al., 1995) and PML bodies (Weis et al., 1994;Dyck et al., 1994;Koken et al., 1994). However, relatively little is known about how distinct nuclear compartments are formed without the use of membranes or about how specific antigens are selectively transported and recruited into these domains. It has been reported that the SR domain, a motif comprised of repeating serine-argine dipeptides found in certain splicing factors, plays a role in subnuclear targeting to speckled structures, though not to coiled bodies (Li and Bingham, 1991 ing complex signals rather than simple peptide motifs (for review see Scheer and Weisenberger, 1994). Although it is still at an early stage, there is increasing in...

Coilin Shuttles between the Nucleus and Cytoplasm InXenopusOocytes

“…For instance, when overexpressed in the oocyte, coilin accumulated in nucleoli unpublished experiments of Zheng'an Wu with green fluorescent protein-tagged coilin). Coilin is also found clustered around nucleoli in HeLa cells treated with actinomycin D (Carmo-Fonseca et al, 1992), and certain mutated forms of coilin appeared in the nucleoli of transfected cells (Bohmann et al, 1995). Taken together, these data suggest that coilin and Nopp140, perhaps in association with fibrillarin and NO38, are part of a transport system involving the cytoplasm, nucleoli, and coiled bodies.…”

“…The primary sequence of human p80-coilin displays both simple (residues 107-112) and bipartite (residues 181-198) NLSs, which are involved in the nuclear import of coilin in transfected cells (Bohmann et al, 1995). These two NLSs are conserved in Xenopus coilin, and because they are potential targets for the importin receptors (Gö rlich, 1998;Ohno et al, 1998), it is likely that nuclear import of coilin requires energy.…”

“…To demonstrate that coilin shuttles between the GV and the cytoplasm, it must be shown that import of anti-coilin antibody does not depend on synthesis of new coilin in the cytoplasm, which is known from previous experiments to be targeted to coiled bodies in the GV and in somatic nuclei (Tuma et al, 1993;Wu et al, 1994;Bohmann et al, 1995). Oocytes were incubated in CHX for 3 h, injected with anti-coilin antibody, and returned to CHX.…”

“…These observations demonstrate that 1) in the GV, an anti-coilin antibody can form an immune complex with coilin in the matrix of the coiled bodies; and 2) an anti-coilin antibody can be imported from the cytoplasm into the GV, where it localizes in the same pattern as endogenous coilin (Tuma et al, 1993;Wu et al, 1994). Because antibodies do not normally cross the nuclear envelope (Bonner, 1975;Einck and Bustin, 1984;Stacey and Allfrey, 1984), the probable interpretation is that the injected antibody binds to coilin in the cytoplasm and is targeted as an antigen-antibody complex to the coiled bodies in the GV.To demonstrate that coilin shuttles between the GV and the cytoplasm, it must be shown that import of anti-coilin antibody does not depend on synthesis of new coilin in the cytoplasm, which is known from previous experiments to be targeted to coiled bodies in the GV and in somatic nuclei (Tuma et al, 1993;Wu et al, 1994;Bohmann et al, 1995). Oocytes were incubated in CHX for 3 h, injected with anti-coilin antibody, and returned to CHX.…”

Targeting SMN to Cajal bodies and nuclear gems during neuritogenesis

“…After several washes, cells were mounted in Vectashield medium (Vector Laboratories). Primary antibodies used were anti-coilin 204.10 rabbit serum (Bohmann et al 1995), anti-SMN monoclonal antibody (mAb) (Transduction Laboratories), anti-SMN 2B1 mAb (Liu and Dreyfuss 1996), anti-Gemin2/SIP1 E17 mAb (Liu et al 1997), anti-Sm C45 human serum, anti-U2B" 4G3 mAb, and anti-Nopp 140 rabbit serum RF12 (Meier and Blobel 1992). Cell samples were examined using a Zeiss 63× NA 1.4 PlanApo objective.…”