Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 May 27;100(11):6475-80.
doi: 10.1073/pnas.1131933100. Epub 2003 May 12.

An actin-ribonucleoprotein interaction is involved in transcription by RNA polymerase II

Piergiorgio Percipalle  1 , Nathalie Fomproix, Karin Kylberg, Francesc Miralles, Birgitta Bjorkroth, Bertil Daneholt, Neus Visa
Affiliations

An actin-ribonucleoprotein interaction is involved in transcription by RNA polymerase II

Piergiorgio Percipalle et al. Proc Natl Acad Sci U S A. .

Abstract

To determine the function of actin in the cell nucleus, we sought to identify nuclear actin-binding proteins in the dipteran Chironomus tentans using DNase I-affinity chromatography. We identified the RNA-binding protein hrp65 as an actin-binding protein and showed that the C-terminal sequence of the hrp65-2 isoform is able to interact directly with actin in vitro. In vivo crosslinking and coimmunoprecipitation experiments indicated that hrp65 and actin are also associated in the living cell. Moreover, in vivo administration of a competing peptide corresponding to the C-terminal sequence of hrp65-2 disrupted the actin-hrp65-2 interaction and caused a specific and drastic reduction of transcription as judged by puff regression and diminished bromo-UTP incorporation. Our results indicate that an actin-based mechanism is implicated in the transcription of most if not all RNA polymerase II genes and suggest that an actin-hrp65-2 interaction is required to maintain the normal transcriptional activity of the cell. Furthermore, immunoelectron microscopy experiments and nuclear run-on assays suggest that the actin-hrp65-2 complex plays a role in transcription elongation.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Actin binds directly to the C terminus of Hrp65-2. (A) Nuclear actin-associated proteins studied by DNase I affinity chromatography and Western blotting. Nuclear extracts from C. tentans tissue-culture cells were either fractionated by SDS/PAGE (N) or mixed with DNase I-Sepharose beads. The proteins in the DNase I-bound fraction (B) were eluted and separated by SDS/PAGE. Gels were silver-stained (lanes 1 and 2) or analyzed by Western blotting by using mAbs against three C. tentans proteins: hrp23 (lanes 3 and 4), hrp36 (lanes 5 and 6), and hrp65 (lanes 7 and 8). The actin band previously identified by mass spectrometry is marked with a dot. (B) Reconstitution experiments with purified [35S]hrp65-2 immobilized on T7 beads and saturating amounts of 35S-labeled G-actin. When indicated, binding was carried out in the presence of either nonconjugated KLH (lane 2) or 65-2CTS-conjugated KLH (lane 3) to compete binding of actin to hrp65-2. The bound proteins were separated by SDS/PAGE and analyzed by autoradiography. (C) Sequences of the peptides used in binding studies. (D) Specificity of the actin–hrp65-2 association assayed by affinity chromatography. The peptides shown in C were coupled to Sulfolink beads via their terminal cysteines and incubated with purified 35S-labeled G-actin. Bound actin was fractionated by SDS/PAGE and visualized by autoradiography. The bound actin was quantified by phosphorimaging, and the results are shown under each lane as percentages of input actin.
Fig. 2.
Fig. 2.
Actin binds directly to Hrp65 in vivo: coimmunoprecipitation after in vivo crosslinking. C. tentans cells were treated with the cell-permeable crosslinker DSP. Nuclear extracts were prepared and split into two equal portions; one was treated with urea. Nuclear extracts were also prepared from DSP-untreated cells (lanes 1 and 2). All samples were subjected to immunoprecipitation with mAb 4E9 against hrp65. Bound proteins were analyzed by SDS/PAGE and Western blotting with rabbit polyclonal antibodies against hrp65 and actin. In some experiments, the cells were incubated in the presence of peptides, either 65-2CTS (lanes 5 and 6) or 118B (lanes 7 and 8), and then treated as described above.
Fig. 3.
Fig. 3.
Disruption of the actin–Hrp65-2 complex down-regulates mRNA transcription. (a–d) Salivary glands were isolated from C. tentans fourth instar larvae, immunostained with mAb 2E4 against hrp45, and visualized by confocal microscopy. Individual salivary gland cells untreated (a), treated with the transcription inhibitor actinomycin D before immunostaining (b), injected with peptide 65-2CTS before immunostaining (c), or injected with control peptides (either 118B or 65-2mut1) before immunostaining (d) are shown. After peptide injections, the glands were incubated in hemolymph at 18°C for 90 min before fixation and immunostaining. For each treatment, three examples are presented: one showing a full nucleus and two additional ones showing only the BR puffs. (e–h) Detection of nascent transcripts by incorporation of BrUTP. BrUTP was injected into the cytoplasm of salivary gland cells, and the incorporation of BrUTP into nascent RNA was visualized by immunofluorescence after 20 min of incubation in hemolymph at 18°C. (e) Incorporation of BrUTP in untreated cells. (f) Glands were incubated with hemolymph containing actinomycin D before BrUTP injection and immunostaining. (g and h) Either peptide 65-2CTS or a control peptide (65-2mut-1 or 118B) was injected into the nucleus of the salivary gland cells, the glands were incubated in hemolymph for 60 min at 18°C, and BrUTP was administered by cytoplasmic injection. The glands then were incubated for an additional 20 min before fixation and immunostaining as described above. (Bar in a, 10 μm.) All images are at the same magnification. The arrowheads point at the BR1 and BR2 puffs in each nucleus.
Fig. 4.
Fig. 4.
Hrp65-2 is associated with the nascent BR pre-mRNP particles: Immunoelectron microscopic localization of hrp65-2 in the active BR transcription unit. Polytene chromosomes were isolated, incubated with the anti-hrp65-2 antibody, and detected with a secondary antibody conjugated to colloidal gold markers. The immunolabeled chromosomes were fixed, dehydrated, embedded in agar resin, and sectioned. (A) Distribution of gold markers in the proximal (p), middle (m), and distal (d) portions of the BR gene. (B) Schematic representation of the active BR transcription unit. (C–H) Examples of immunolabeling in nascent BR pre-mRNP particles. Interpretations of the images are provided under each micrograph. (Bar, 100 nm.)
See this image and copyright information in PMC

References

    1. Rando, O. J., Zhao, K. & Crabtree, G. R. (2000) Trends Cell Biol. 10, 92–97. - PubMed
    1. Pestic-Dragovich, L., Stojiljkovic, L., Philimonenko, A. A., Nowak, G., Ke, Y., Settlage, R. E., Shabanowitz, J., Hunt, D. F., Hozak, P. & deLanerolle, P. (2000) Science 290, 337–341. - PubMed
    1. Scheer, U., Hinssen, H., Franke, W. W. & Jockusch, B. M. (1984) Cell 39, 111–122. - PubMed
    1. Zhao, K., Wang, W., Rando, O. J., Xue, Y., Swiderek, K., Kuo, A. & Crabtree, G. R. (1998) Cell 95, 625–636. - PubMed
    1. Sotiropoulos, A., Gineitis, D., Copeland, J. & Treisman, R. (1999) Cell 98, 159–169. - PubMed

Publication types

LinkOut - more resources