Presence of a Unique Set of Nuclear Proteins at the Sites Sensitive to RNase A as Well as DNase I

Inoue, Akira; Higashi, Youtaro; Hasuma, Tadayoshi; Yukioka, Munehiko; Morisawa, Seiji

doi:10.1093/oxfordjournals.jbchem.a121767

Cited by 11 publications

(8 citation statements)

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“…S1 proteins C2 and D2 were novel hnRNP proteins. The present results are consistent with the previous observations: S1 proteins occurred at sites highly sensitive to DNase I as well as RNase A in the cell nucleus [4,5]. It is comprehended that RNase A digests the S1 protein‐containing nascent RNPs, and releases the fragments to the supernatants; likewise, DNase I rapidly degrades and liberates transcriptionally active chromatin as small fragments [44], which must contain nascent RNPs as well as transcription factors.…”

Section: Discussionsupporting

confidence: 92%

“…They are present at sites sensitive to RNase as well as DNase, and extracted at pH 4.9 from the reaction mixture of nuclei treated mildly with either enzyme. Under these conditions, most proteins liberated from the nuclei become aggregated, and S1 proteins are left behind in the supernatant S1 (the first supernatant termed in the original experiments) [5,6]. This protein‐aggregation is largely due to isoelectric precipitation at or near the isoelectric points of associated RNPs and chromatin (Y. Fujitsuka, K. Tsugawa & A. Inoue, unpublished results).…”

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confidence: 99%

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S1 proteins C2 and D2 are novel hnRNPs similar to the transcriptional repressor, CArG box motif‐binding factor A

Inoue

Omori

Ichinose

et al. 2001

European Journal of Biochemistry

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S1 proteins A±D are liberated from thoroughly washed nuclei by mild digestion with DNase I or RNase A, and extracted selectively at pH 4.9 from the reaction supernatants. Here, we characterized the S1 proteins, focusing on protein D2, the most abundant S1 protein in the rat liver, and on protein C2 as well. Using a specific antibody, McAb 351, they were shown to occur in the extranucleolar nucleoplasm, and to be extracted partly in the nuclear soluble fraction. We demonstrate that the S1 proteins in this fraction exist constituting heterogeneous nuclear ribonucleoproteins (hnRNPs), through direct binding to hnRNAs, as revealed by centrifugation on density gradients, immunoprecipitation, and UV cross-linking. In hnRNPs, protein D2 occurred at nuclease-hypersensitive sites and C2 in the structures that gave rise to 40 S RNP particles. By microsequencing, protein D2 was identified with a known protein, CArG box motif-binding factor A (CBF-A), which has been characterized as a transcriptional repressor, and C2 as its isoform protein. In fact, CBF-A expressed from its cDNA was indistinguishable from protein D2 in molecular size and immunoreactivity to McAb 351. Thus, the present results demonstrate that S1 proteins C2 and D2 are novel hnRNP proteins, and suggest that the proteins C2 and D2 act in both transcriptional and post-transcriptional processes in gene expression.

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Section: Discussionsupporting

confidence: 92%

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confidence: 99%

S1 proteins C2 and D2 are novel hnRNPs similar to the transcriptional repressor, CArG box motif‐binding factor A

Inoue

Omori

Ichinose

et al. 2001

European Journal of Biochemistry

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“…Purified nuclei were prepared from tissues of various rat organs, as described in [21], by centrifugation at 50000 x g for 90 min through 2.3 M sucrose/3 mM MgCI2/0.2 mM phenylmethylsulfonyl fluoride, in 4.5 (brain), 7 (heart), 8 (spleen and testis), and 9 (liver and kidney) final tissue volumes. The pelleted nuclei were washed three times in buffer A and used for the T3-binding assay, or for solubilization of T3-receptor protein.…”

Section: Isolation Ojnuclei From Rat Tissuesmentioning

confidence: 99%

Unesterified long‐chain fatty acids inhibit thyroid hormone binding to the nuclear receptor

Inoue

Yamamoto

Morisawa

et al. 1989

European Journal of Biochemistry

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Unesterified long-chain fatty acids strongly inhibited thyroid hormone (T3) binding to nuclear receptors extracted from rat liver, kidney, spleen, brain, testis and heart. Oleic acid was the most potent inhibitor, attaining 50% inhibition at 2.8 pM. Oleic acid similarly inhibited the partially purified receptor and enhanced dissociation of the preformed T3-receptor complex. The fatty acid acted in a soluble form and in a competitive manner for the T,-binding sites, thereby reducing the affinity of the receptor for T,. The affinity of the receptor for oleic acid (Ki) was 1.0 pM. In HTC rat hepatoma cells in culture, fatty acids added to the medium reached the nucleus and inhibited nuclear T3 binding; oleic acid being the most potent. T3 binding of the cells was reversibly restored in fresh medium free of added fatty acids. Oleic acid did not affect all the T3-binding sites in the HTC cells: one form (80%) was inhibited and the other was not and these two forms were commonly present in all rat tissues examined. Thus, fatty acids inhibited the solubilized nuclear receptor as well as a class of nuclear T3-binding sites in cells in culture.Specific nuclear receptors for thyroid hormone (triiodothyronine; T3) exist in association with chromatin and participate in the regulation of transcription [l]. T,-responsive elements have been identified in the 5'4anking regions of rat and human growth hormone genes [2 -51. Other examples are the induction by T3 of the or-myosin heavy-chain gene [6, 71, and repression of thyrotropin a-and p-chain genes [8]. The hormone acts also at post-transcriptional steps [9 -111, such as in the stabilization of nuclear mRNA precursors [12]. In addition to the chromatin-associated receptor, other T3-binding sites are present in the soluble cytoplasm, plasma membrane, mitochondria, endoplasmic reticulum and nuclear envelope [33, 141. The chromatin receptor has been separated from other T3 binders, essentially by isolation of nuclei, followed by repeated washing in a solution containing a detergent such as 3-[3-(chloramidopropy1)-dimethylammonio]-1-propane sulfate (Chaps) or Triton X-100, to remove cytoplasmic contaminants and nuclear-envelope-associated binders [14-171.In early work we found that when the solubilized nuclear T3 receptor was treated with a lipase, the T,-binding activity of the receptor was significantly reduced, These events proved to be linked to fatty acids, presumably generated by hydrolysis of lipids present in the solubilized receptor preparation (unpublished observation).In the present study, we show that unesterified long-chain fatty acids inhibited T3 binding by the nuclear receptor solubilized from the chromatin of various rat organs, and also T3 binding in HTC rat hepatoma cells in culture. There were two forms of binding sites, in terms of sensitivity to inhibition in the cultured cells as well as in the isolated nuclei: one was inhibited by fatty acids and designated here as the responder, and the other was not inhibited and was designated the nonresponder. During p...

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“…This suggests hnRNP A/B associates with speckles/IGCs in an RNA-dependent manner. These proteins occur at sites that are highly sensitive to RNase in the nuclei and are rapidly released after treatment with RNase, which digests protein-containing nascent RNPs (Inoue et al 1986;Amero et al 1993).…”

Section: Discussionmentioning

confidence: 99%

Localization of poly(A)+ RNA and mRNA export factors in interchromatin granule clusters of two-cell mouse embryos

2009

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Interchromatin granule clusters (IGCs), also known as nuclear speckles, splicing factor compartments, or SC35-domains, are one of the most universal nuclear organelles of the cell. We have used two-cell mouse embryos as an experimental system to study the possible association of poly(A)(+) RNA and factors involved in RNA export (Tip-associated protein [TAP] and heterogenous nuclear ribonucleoprotein A/B [hnRNP A/B]) with IGCs. Poly(A)(+) RNA was localized by microinjections of 2'-O-Me(U)(22) probes conjugated with tetramethylrhodamine. RNA export proteins were detected by immunofluorescence confocal laser microscopy and immunogold-labeling electron microscopy. We found that poly(A)(+) RNA was located in IGCs regardless of the transcriptional state of the nuclei. hnRNP A/B was also found to characterize IGCs of the embryo nuclei with various levels of transcription activity. In transcriptionally active embryo nuclei, TAP was detected in the vicinity of IGCs rather than inside them; however, when transcription was inhibited by drugs, TAP was localized to IGCs. Our data support the idea that IGCs not only serve as splicing factor reservoirs, but also take part in mRNA retention and export.

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Presence of a Unique Set of Nuclear Proteins at the Sites Sensitive to RNase A as Well as DNase I

Cited by 11 publications

References 0 publications

S1 proteins C2 and D2 are novel hnRNPs similar to the transcriptional repressor, CArG box motif‐binding factor A

S1 proteins C2 and D2 are novel hnRNPs similar to the transcriptional repressor, CArG box motif‐binding factor A

Unesterified long‐chain fatty acids inhibit thyroid hormone binding to the nuclear receptor

Localization of poly(A)+ RNA and mRNA export factors in interchromatin granule clusters of two-cell mouse embryos

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