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. 2024 Mar;300(3):105698.
doi: 10.1016/j.jbc.2024.105698. Epub 2024 Jan 30.

Actin associates with actively elongating genes and binds directly to the Cdk9 subunit of P-TEFb

Salla Kyheröinen  1 , Bina Prajapati  1 , Maria Sokolova  1 , Maximilian Schmitz  2 , Tiina Viita  1 , Matthias Geyer  2 , Maria K Vartiainen  3
Affiliations

Actin associates with actively elongating genes and binds directly to the Cdk9 subunit of P-TEFb

Salla Kyheröinen et al. J Biol Chem. 2024 Mar.

Abstract

Nuclear actin has been demonstrated to be essential for optimal transcription, but the molecular mechanisms and direct binding partner for actin in the RNA polymerase complex have remained unknown. By using purified proteins in a variety of biochemical assays, we demonstrate a direct and specific interaction between monomeric actin and Cdk9, the kinase subunit of the positive transcription elongation factor b required for RNA polymerase II pause-release. This interaction efficiently prevents actin polymerization, is not dependent on kinase activity of Cdk9, and is not involved with releasing positive transcription elongation factor b from its inhibitor 7SK snRNP complex. Supporting the specific role for actin in the elongation phase of transcription, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) reveals that actin interacts with genes only upon their active transcription elongation. This study therefore provides novel insights into the mechanisms by which actin facilitates the transcription process.

Keywords: Cdk9; P-TEFb; RNA polymerase II; actin; nucleus; transcription.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Actin associates with hsp genes only upon their active transcription elongation.A, actin binds hsp genes in Drosophila ovaries. Normalized (RPKM) coverage of RNA polymerase phosphorylated on serine 5 (Pol2S5P), actin, and normal IgG from chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) on Hsp68 in Drosophila ovaries (W1118). B, Pol II S5P, actin, and mouse IgG coverage on 15 heat shock responding genes in Drosophila ovaries. Normalized fragment counts are scaled to 5 kb gene size. Transcription start site (TSS) and cleavage and polyadenylation site (CPS). C, actin binds to hsp genes only upon their transcriptional activation during heat shock (HS). Normalized (RPKM) coverage of Pol II S5P, actin, and normal IgG on Hsp68 from ChIP-seq in S2R+ cells in non heat shock (NHS) and HS (20 min) conditions. D, Pol II S5P coverage on 15 HS responding genes in S2R+ cells in NHS and HS conditions. Input is used as a negative control and data shown as in (B). E, actin coverage on 15 HS responding genes in S2R+ cells in NHS and HS conditions, shown as in (D).
Figure 2
Figure 2
Cdk9 binds directly to G-actin with a preference towards ATP-actin.A, Western blot of co-immunoprecipitation (IP) experiment for 2Flag-Cdk9 (prey) and 2HA-actin (bait), using empty HA as a control. B, Western blot of co-IP experiment for 2HA-Cdk9 (prey) and indicated Flag/2Flag-tagged actin mutants (baits). Boxed areas cut from the same exposure to remove a construct with weak expression level and a lane with protein ladder. C, Western blot of pulldown assay of LatB-actin using His-Cdk9 as the bait and His-GST as a control bait. His-GST detected with His-HRP antibody, His-Cdk9 with Cdk9 antibody, and LatB-actin with Ac-40 actin antibody. D, NBD-actin monomer-binding assay showing increase in the fluorescence of 0.2 μM NBD-labeled ATP-Mg-G-actin under physiological ionic conditions over a range of concentrations of Cdk9 or known actin-binding protein cofilin. Symbols are data points and lines represent the exponentially fitted binding curves. N = 3, one example presented. Experimentally determined dissociation constants (KD) calculated according to formula presented in materials and methods. Average KD of three assays is 375 ± 105 nM. E, NBD-actin monomer-binding assay as in (A), but using ADP-bound actin. LatB, Latrunculin B.
Figure 3
Figure 3
Actin binds to P-TEFb via Cdk9 and the binding inhibits actin polymerization. Pyrene actin polymerization assay with (A) P-TEFb, (B) Cdk9, (C) Cyclin T1, (D) Cdk12/Cyclin K, (E) Cdk7/Cyclin H/Mat1 added in increasing concentrations and actin polymerization monitored by increase in the pyrene fluorescence. Actin concentration 4 μM, except for in the experiment with Cdk9 where actin is 2.5 μM. An example graph from one assay is presented. P-TEFb, positive transcription elongation factor b.
Figure 4
Figure 4
Kinase activity of Cdk9 is not required for the interaction with actin or actin recruitment to hsp genes.A, Pyrene actin polymerization assay with Cdk9 (0 and 5 μM) and Cdk9 inhibitors. Flavopiridol was 15 μM, NVP-2 5 μM, and iCdk9 10 μM. Actin concentration, 2.5 μM. B, chromatin immunoprecipitation with actin Ac-74 antibody followed by RT-qPCR (ChIP-qPCR) of hsp70 middle region (Hsp70mid) in Drosophila S2R+ cells. Cells were treated with DMSO/Flavopiridol for 10 min, followed by 20 min heat shock and immediate fixing. N = 3, error bars ± 0.5 SD. Data presented as % of input enrichment. Statistical significance tested with two-sample t test, DMSO versus Flavopiridol p = 0.130.
Figure 5
Figure 5
In vitro, actin does not influence P-TEFb activity.A, Western blot of P-TEFb release from HEXIM1 immunoprecipitation by RNAse A and increasing amounts of purified rabbit muscle actin (+0.5 μM and ++ 2 μM). P-TEFb probed with Cdk9 antibody and HEXIM1 with HEXIM1 antibody. In control sample (first lane), no release agent added. Boxed areas cut from the same exposure to remove an extra control sample. B, in vitro kinase assay assessing recombinant P-TEFb kinase activity for phosphorylation of CTD52 substrate, and without the substrate as a control, with increasing LatB-actin amounts added to the reaction. Mean of triplicate measurements is presented, error bars ± 0.5 SD. ∗Statistically significant differences (p < 0.05) with a two-sample t test for samples with CTD substrate: 0 versus 0.2 μM actin p = 0.532, 0 versus. 1 μM actin p = 0.730, 0 versus. 4 μM actin p = 0.002. P-TEFb, positive transcription elongation factor b; LatB, Latrunculin B.
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References

    1. Ulferts S., Prajapati B., Grosse R., Vartiainen M.K. Emerging properties and functions of actin and actin filaments inside the nucleus. Cold Spring Harb. Perspect. Biol. 2021;13:a040121. - PMC - PubMed
    1. Dopie J., Skarp K.P., Kaisa Rajakyla E., Tanhuanpaa K., Vartiainen M.K. Active maintenance of nuclear actin by importin 9 supports transcription. Proc. Natl. Acad. Sci. U. S. A. 2012;109:E544–E552. - PMC - PubMed
    1. Stuven T., Hartmann E., Gorlich D. Exportin 6: a novel nuclear export receptor that is specific for profilin.actin complexes. EMBO J. 2003;22:5928–5940. - PMC - PubMed
    1. Klages-Mundt N.L., Kumar A., Zhang Y., Kapoor P., Shen X. The nature of actin-family proteins in chromatin-modifying complexes. Front. Genet. 2018;9:398. - PMC - PubMed
    1. Vartiainen M.K., Guettler S., Larijani B., Treisman R. Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL. Science. 2007;316:1749–1752. - PubMed

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