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Macromolecular domains containing nuclear protein p107 and U-snRNP protein p28: Further evidence for an in situ nuclear matrix

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Summary

Polyclonal antibodies have been produced which react with a nuclear protein having a molecular weight of 107kD and a pl of 8.7–8.8 (designated p107). This protein is shown to be a component of the residual ribonucleoprotein (RNP) network of the nuclear matrix. P107 localized exclusively to the nuclear interior but not within nucleolar or chromatin domains. We have taken advantage of this unique probe to examine whether the RNP network of the isolated nuclear matrix has a physical counterpart in situ. We show that RNA, p107, divalent cations and the 28 kD Sm antigen of U-snRNPs are components of in situ macromolecular assemblies. While the morphology and intranuclear distribution of these assemblies are insensitive to the removal of chromatin, they are markedly altered by degradation of RNA. Digestion in situ of RNA in the presence of EDTA followed by extraction with high ionic strength buffers solubilized the components of these assemblies. Electron microscopic and immunobiochemical data are presented which support the concept that the residual RNP network of the nuclear matrix is an isolate of a pre-existing structure, and that perturbations in this internal network can be created by RNA degradation, depletion of essential metal ions and proteolysis.

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Abbreviations

CRLM:

polyclonal chicken antibody raised against rat liver nuclear matrix

Sm:

monoclonal antibody specific for the 28 kd protein antigen of U1, U2, U4, U5 and U6 snRNPs

hnRNP:

ribonucleoprotein particles containing hnRNA

snRNP:

ribonucleoprotein particles containing snRNA

PBS:

phosphate buffered saline

PMSF:

phenylmethylsulfonyl fluoride

PAGE:

polyacrylamide gel electrophoresis

EDTA:

ethylenediaminetetraacetic acid

VRC:

vanadium ribonucleoside complex

BSA:

bovine serum albumin

DMSO:

dimethylsulfoxide

HS:

high salt buffer

LS:

low salt buffer

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Author notes

  1. Harold C. Smith

    Present address: Department of Medicine, Howard Hughes Medical Institute, Baylor College of Medicine, 77030, Houston, TX, USA

  2. David L. Spector

    Present address: Cold Spring Harbor Laboratory, P.O. Box 100, 11724, Cold Spring Harbor, NY, USA

Authors and Affiliations

  1. Department of Pharmacology, Baylor College of Medicine, 77030, Houston, TX, USA

    Harold C. Smith, Robert L. Ochs, Enrique A. Fernandez & David L. Spector

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  1. Harold C. Smith
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Smith, H.C., Ochs, R.L., Fernandez, E.A. et al. Macromolecular domains containing nuclear protein p107 and U-snRNP protein p28: Further evidence for an in situ nuclear matrix. Mol Cell Biochem 70, 151–168 (1986). https://doi.org/10.1007/BF00229430

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