Intermolecular recognition of the non-coding RNA 7SK and HEXIM protein in perspective

Martinez-Zapien, Denise; Saliou, Jean‐Michel; Han, Xiao; Atmanène, Cédric; Proux, Florence; Cianférani, Sarah; Dock-Brégeon, Anne-Catherine

doi:10.1016/j.biochi.2015.03.020

Cited by 18 publications

(23 citation statements)

References 56 publications

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Section: Introductionmentioning

confidence: 99%

“…Moreover, although it is a symmetrical sequence, it binds only one ARM-motif of a HEXIM dimer (26,28). What the second ARM does is still an open issue, but it probably has consequences on the functional mechanism converting HEXIM into a P-TEFb inhibitor (28). It is expected that this mechanism relies on conformational changes of 7SK upon HEXIM binding (29), thus making the highly flexible and modular nature of the 7SK RNA a functional requirement.…”

Section: Introductionmentioning

confidence: 99%

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The crystal structure of the 5′ functional domain of the transcription riboregulator 7SK

et al. 2017

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In vertebrates, the 7SK RNA forms the scaffold of a complex, which regulates transcription pausing of RNA-polymerase II. By binding to the HEXIM protein, the complex comprising proteins LARP7 and MePCE captures the positive transcription elongation factor P-TEFb and prevents phosphorylation of pausing factors. The HEXIM-binding site embedded in the 5΄-hairpin of 7SK (HP1) encompasses a short signature sequence, a GAUC repeat framed by single-stranded uridines. The present crystal structure of HP1 shows a remarkably straight helical stack involving several unexpected triples formed at a central region. Surprisingly, two uridines of the signature sequence make triple interactions in the major groove of the (GAUC)2. The third uridine is turned outwards or inward, wedging between the other uridines, thus filling the major groove. A molecular dynamics simulation indicates that these two conformations of the signature sequence represent stable alternatives. Analyses of the interaction with the HEXIM protein confirm the importance of the triple interactions at the signature sequence. Altogether, the present structural analysis of 7SK HP1 highlights an original mechanism of swapping bases, which could represent a possible ‘7SK signature’ and provides new insight into the functional importance of the plasticity of RNA.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The crystal structure of the 5′ functional domain of the transcription riboregulator 7SK

et al. 2017

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“…P-TEFb retained on Strep-Tactin Sepharose beads (IBA) was eluted with 2.5 mM desthiobiotin. 7SK RNA was prepared by T7 polymerase in vitro transcription (52). In vitro P-TEFb.Hexim1.7SK RNA complex reconstitution and cross-linking.…”

Section: Methodsmentioning

confidence: 99%

An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb

Kobbi

Demey-Thomas

Brayé

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The positive transcription elongation factor (P-TEFb) is required for the transcription of most genes by RNA polymerase II. Hexim proteins associated with 7SK RNA bind to P-TEFb and reversibly inhibit its activity. P-TEFb comprises the Cdk9 cyclin-dependent kinase and a cyclin T. Hexim proteins have been shown to bind the cyclin T subunit of P-TEFb. How this binding leads to inhibition of the kinase activity of Cdk9 has remained elusive, however. Using a photoreactive amino acid incorporated into proteins, we show that in live cells, cell extracts, and in vitro reconstituted complexes, Hexim1 cross-links and thus contacts Cdk9. Notably, replacement of a phenylalanine, F208, belonging to an evolutionary conserved Hexim1 peptide ( 202 PYNTTQFLM 210 ) known as the "PYNT" sequence, cross-links a peptide within the activation segment that controls access to the Cdk9 catalytic cleft. Reciprocally, Hexim1 is cross-linked by a photoreactive amino acid replacing Cdk9 W193, a tryptophan within this activation segment. These findings provide evidence of a direct interaction between Cdk9 and its inhibitor, Hexim1. Based on similarities with Cdk2 3D structure, the Cdk9 peptide cross-linked by Hexim1 corresponds to the substrate bindingsite. Accordingly, the Hexim1 PYNT sequence is proposed to interfere with substrate binding to Cdk9 and thereby to inhibit its kinase activity.cyclin-dependent kinase inhibition | transcription factor regulation | regulatory noncoding RNA | benzoyl phenylalanine | protein-protein cross-linking

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“…Previous work suggested that Hexim binds 7SK as a dimer Dulac et al 2005;Li et al 2005). The way Hexim interacts with the 7SK RNA is still an open question, and several possible binding mechanisms have been proposed (Martinez-Zapien et al 2015). Among them, the repeated GAUC-GAUC bordered by U-bulges (motif M2) may provide a high-affinity primary binding site targeted by the ARM motif of Hexim, and the M1 region may provide a secondary weak binding site contacted by another part of the protein.…”

Section: The Recognition Of Hpi Could Be Driven By a Conformational Smentioning

confidence: 99%

“…More recently, an alternative secondary structure based on computational analysis has been proposed, with major differences in the middle part of the 7SK RNA while the upper part of HPI and HP4 structures were conserved (Marz et al 2009). Several studies demonstrated that the 5 ′ -terminal hairpin of 7SK includes the Hexim1 binding site, the (24-87) region being the minimal sequence required for interaction (Egloff et al 2006;Belanger et al 2009;Martinez-Zapien et al 2015). The 3 ′ -terminal hairpin was also identified as essential for P-TEFb regulation but not involved in Hexim1 binding (Muniz et al 2013).…”

Section: Introductionmentioning

confidence: 99%

Solution structure of the 5′-terminal hairpin of the 7SK small nuclear RNA

Bourbigot¹,

Dock-Brégeon²,

Eberling³

et al. 2016

RNA

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The small nuclear 7SK RNA regulates RNA polymerase II (RNA Pol II) transcription, by sequestering and inhibiting the positive transcription elongation factor b (P-TEFb). P-TEFb is stored in the 7SK ribonucleoprotein (RNP) that contains the three nuclear proteins Hexim1, LaRP7, and MePCE. P-TEFb interacts with the protein Hexim1 and the 7SK RNA. Once P-TEFb is released from the 7SK RNP, it activates transcription by phosphorylating the C-terminal domain of RNA Pol II. P-TEFb also plays a crucial role in the replication of the human immunodeficiency virus HIV-1, through its recruitment by the viral transactivator Tat. Previous work demonstrated that the protein Tat promotes the release of P-TEFb from the 7SK RNP through direct binding to the 7SK RNA. Hexim1 and Tat proteins both comprise conserved and similar arginine-rich motifs that were identified to bind the 7SK RNA at a repeated GAUC site located at the top of the 5 ′ -terminal hairpin (HPI). Here, we report the solution structure of this region as determined by nuclear magnetic resonance, to identify HPI structural features recognized by Hexim1 and Tat. The HPI solution structure displays an elongated shape featuring four helical segments interrupted by one internal loop and three bulges with distinct folds. In particular, the repeated GAUC motif adopts a pre-organized geometry. Our results suggest that the binding of Hexim1 and Tat to the 7SK RNA could originate from a conformational selection of this motif, highlighting how RNA local structure could lead to an adaptive recognition of their partners.

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Intermolecular recognition of the non-coding RNA 7SK and HEXIM protein in perspective

Cited by 18 publications

References 56 publications

The crystal structure of the 5′ functional domain of the transcription riboregulator 7SK

The crystal structure of the 5′ functional domain of the transcription riboregulator 7SK

An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb

Solution structure of the 5′-terminal hairpin of the 7SK small nuclear RNA

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