Interplay between 7SK snRNA and oppositely charged regions in HEXIM1 direct the inhibition of P‐TEFb

Transcription elongation of eukaryotic genes by RNA polymerase II depends on the positive transcription elongation factor b (P‐TEFb). When sequestered into the large complex, P‐TEFb kinase activity is inhibited by the coordinate actions of 7SK small nuclear RNA (7SK snRNA) and hexamethylene bisacetamide (HMBA)‐induced protein 1 (HEXIM1). We found that the basic region in HEXIM1 directs its nuclear import via two monopartite and two bipartite nuclear localization sequences. Moreover, the arginine‐rich motif within it is essential for its binding to 7SK snRNA, P‐TEFb, and inhibition of transcription. Notably, the basic region interacts with the adjacent acidic regions in the absence of RNA. The removal of the positive or negative charges from these regions in HEXIM1 leads to its sequestration into the large complex and inhibition of transcription independently of the arginine‐rich motif. Finally, the removal of the negative charges from HEXIM1 results in its subnuclear localization into nuclear speckles. We propose a model where the interplay between 7SK snRNA and oppositely charged regions in HEXIM1 direct its binding to P‐TEFb and subcellular localization that culminates in the inhibition of transcription.

[1]  O. Bensaude,et al.  MAQ1 and 7SK RNA Interact with CDK9/Cyclin T Complexes in a Transcription-Dependent Manner , 2003, Molecular and Cellular Biology.

[2]  B. Peterlin,et al.  Identification of a Cyclin T-Binding Domain in Hexim1 and Biochemical Analysis of Its Binding Competition with HIV-1 Tat* , 2005, Journal of Biological Chemistry.

[3]  D. Price,et al.  Flavopiridol Inactivates P-TEFb and Blocks Most RNA Polymerase II Transcription in Vivo * , 2001, The Journal of Biological Chemistry.

[4]  M. Mancini,et al.  The Cdk9 and cyclin T subunits of TAK/P-TEFb localize to splicing factor-rich nuclear speckle regions. , 2001, Journal of cell science.

[5]  Qiang Zhou,et al.  Compensatory Contributions of HEXIM1 and HEXIM2 in Maintaining the Balance of Active and Inactive Positive Transcription Elongation Factor b Complexes for Control of Transcription* , 2005, Journal of Biological Chemistry.

[6]  O. Bensaude,et al.  Transcription-dependent Association of Multiple Positive Transcription Elongation Factor Units to a HEXIM Multimer* , 2005, Journal of Biological Chemistry.

[7]  Tamás Kiss,et al.  7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes , 2001, Nature.

[8]  Qiang Zhou,et al.  The 7SK small nuclear RNA inhibits the CDK9/cyclin T1 kinase to control transcription , 2001, Nature.

[9]  Qintong Li,et al.  HEXIM2, a HEXIM1-related Protein, Regulates Positive Transcription Elongation Factor b through Association with 7SK* , 2005, Journal of Biological Chemistry.

[10]  H. Handa,et al.  Suppression of NF‐κB‐dependent gene expression by a hexamethylene bisacetamide‐inducible protein HEXIM1 in human vascular smooth muscle cells , 2003, Genes to cells : devoted to molecular & cellular mechanisms.

[11]  R. van Driel,et al.  Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection. , 1999, Molecular biology of the cell.

[12]  M. Carmo-Fonseca.  The Contribution of Nuclear Compartmentalization to Gene Regulation , 2002, Cell.

[13]  Karsten Weis,et al.  Regulating Access to the Genome Nucleocytoplasmic Transport throughout the Cell Cycle , 2003, Cell.

[14]  B. Peterlin,et al.  Interaction between P-TEFb and the C-Terminal Domain of RNA Polymerase II Activates Transcriptional Elongation from Sites Upstream or Downstream of Target Genes , 2002, Molecular and Cellular Biology.

[15]  A. Link,et al.  Inhibition of P-TEFb (CDK9/Cyclin T) kinase and RNA polymerase II transcription by the coordinated actions of HEXIM1 and 7SK snRNA. , 2003, Molecular cell.

[16]  B. Peterlin,et al.  A New Paradigm in Eukaryotic Biology: HIV Tat and the Control of Transcriptional Elongation , 2005, PLoS biology.

[17]  A. Giordano,et al.  Activation and function of cyclin T–Cdk9 (positive transcription elongation factor-b) in cardiac muscle-cell hypertrophy , 2002, Nature Medicine.

[18]  B. Peterlin,et al.  Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb , 2005, Nucleic acids research.

[19]  Danny Reinberg,et al.  Elongation by RNA polymerase II: the short and long of it. , 2004, Genes & development.

[20]  K. Yamaguchi,et al.  Cloning of Hexamethylene-bis-acetamide-inducible Transcript, HEXIM1, in Human Vascular Smooth Muscle Cells , 1999 .

[21]  David L. Spector,et al.  Nuclear speckles: a model for nuclear organelles , 2003, Nature Reviews Molecular Cell Biology.

[22]  Qiang Zhou,et al.  A Human Immunodeficiency Virus Type 1 Tat-Like Arginine-Rich RNA-Binding Domain Is Essential for HEXIM1 To Inhibit RNA Polymerase II Transcription through 7SK snRNA-Mediated Inactivation of P-TEFb , 2004, Molecular and Cellular Biology.

[23]  Bryan R. Cullen,et al.  The Arginine-Rich Domains Present in Human Immunodeficiency Virus Type 1 Tat and Rev Function as Direct Importin β-Dependent Nuclear Localization Signals , 1999, Molecular and Cellular Biology.

[24]  M. Malim,et al.  Importin β Can Mediate the Nuclear Import of an Arginine-Rich Nuclear Localization Signal in the Absence of Importin α , 1999, Molecular and Cellular Biology.

[25]  Van Trung Nguyen,et al.  Binding of the 7SK snRNA turns the HEXIM1 protein into a P‐TEFb (CDK9/cyclin T) inhibitor , 2004, The EMBO journal.

[26]  M. Lai,et al.  Hepatitis Delta Antigen Mediates the Nuclear Import of Hepatitis Delta Virus RNA , 1998, Journal of Virology.

[27]  Dongmei Cheng,et al.  Analysis of the Large Inactive P-TEFb Complex Indicates That It Contains One 7SK Molecule, a Dimer of HEXIM1 or HEXIM2, and Two P-TEFb Molecules Containing Cdk9 Phosphorylated at Threonine 186* , 2005, Journal of Biological Chemistry.