Piergiorgio Percipalle *, Nathalie Fomproix *, Karin Kylberg *, Francesc Miralles 1 , Birgitta Björkroth *, Bertil Daneholt *, and Neus Visa1, @
*Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, SE-17177 Stockholm, Sweden; and 1 Department of Molecular Biology and Functional Genomics, Stockholm University, SE-10691 Stockholm, Sweden
@ To whom correspondence should be addressed.
E-mail: neus.visa@molbio.su.se
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.
1. Kiesler E, Miralles F, and Visa N, "HEL/UAP56 Binds Cotranscriptionally to the Balbiani Ring pre-mRNA in an Intron-Independent Manner and Accompanies the BR mRNP to the Nuclear Pore", Curr. Biol., vol. 12, no. 10, pp. 859-862 (May 14, 2002).
2. Percipalle P, Jonsson A, Nashchekin D, Karlsson C, Bergman T, Guialis A, and Daneholt B, "Nuclear Actin is Associated with a Specific Subset of hnRNP A/B-type Proteins", Nucleic Acids Res., vol. 30 no. 8, pp. 1725-1734 (April 15, 2002).
3. Frenster JH, Allfrey VG, and Mirsky AE, "Metabolism and Morphology of Ribonucleoprotein Particles from the Cell Nucleus of Lymphocytes", Proc. Natl. Acad. Sci. U.S.A., vol. 46, pp. 432-444 (April, 1960).
4. Hovsepian JA, and Frenster JH, "RNA-Induced Melting of DNA during Selective Gene Transcription", Molec. Biol. Cell, vol. 13, supp. p. 239a (November, 2002).
5. Saha S, Ansari AZ, Jarell KA, and Ptashne M, "RNA Sequences that
Work as Transcriptional Activating Regions", Nucleic
Acid Research, vol. 31, no. 5, pp. 1565-1570 (March 1, 2003).