Preformed Protein-binding Motifs in 7SK snRNA: Structural and Thermodynamic Comparisons with Retroviral TAR

Durney, Michael A.; D'Souza, Ventris M.

doi:10.1016/j.jmb.2010.08.042

Cited by 28 publications

(47 citation statements)

References 40 publications

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“…Their imino protons are broad, which indicate that these residues are in weak stacking and interactions, thus able to adopt other conformations not detectable by NMR. Our observations for M1 and M2 motifs are reminiscent of those reported for the 7SK HP4 RNA hairpin that comprises a dinucleotide CU bulge (Durney and D'Souza 2010). In HP4, the C residue was found to be positioned into the major groove in the proximity of a G:C base pair, although no direct hydrogen bond could be observed.…”

Section: Discussionsupporting

confidence: 79%

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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Section: Discussionsupporting

confidence: 79%

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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“…SL4 is highly conserved in both sequence and secondary structure, with loop residue G312 100% conserved (22). The solution NMR structure of a human 7SK SL4 construct bound to arginine showed that bulge residue C320 forms an unusual base triple with the G303-C323 base pair (23), similar to the base triple observed in HIV TAR (24–26). …”

Section: Introductionmentioning

confidence: 58%

“…These results provide further evidence that the CU bulge is not part of the xRRM binding site, and in fact decreases binding affinity. C320 in the CU bulge was proposed to form a base triple with the G303-C323 base pair in the lower stem in the free SL4 (23); whether this triple forms in the free RNA or when hLARP7 binds under the ITC buffer conditions, and if so, interferes with binding of helix α3x remains to be determined. Together, these data define the binding site for hLARP7 xRRM as the upper stem-loop of SL4 and indicate that the CU bulge does not contribute to specific binding.…”

Section: Resultsmentioning

confidence: 99%

hLARP7 C-terminal domain contains an xRRM that binds the 3′ hairpin of 7SK RNA

2016

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The 7SK small nuclear ribonucleoprotein (snRNP) sequesters and inactivates the positive transcription elongation factor b (P-TEFb), an essential eukaryotic mRNA transcription factor. The human La-related protein group 7 (hLARP7) is a constitutive component of the 7SK snRNP and localizes to the 3′ terminus of the 7SK long noncoding RNA. hLARP7, and in particular its C-terminal domain (CTD), is essential for 7SK RNA stability and assembly with P-TEFb. The hLARP7 N-terminal La module binds and protects the 3′ end from degradation, but the structural and functional role of its CTD is unclear. We report the solution NMR structure of the hLARP7 CTD and show that this domain contains an xRRM, a class of atypical RRM first identified in the Tetrahymena thermophila telomerase LARP7 protein p65. The xRRM binds the 3′ end of 7SK RNA at the top of stem-loop 4 (SL4) and interacts with both unpaired and base-paired nucleotides. This study confirms that the xRRM is general to the LARP7 family of proteins and defines the binding site for hLARP7 on the 7SK RNA, providing insight into function.

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“…This led to a model in which P-TEFb is held inactive by the 7SK snRNP in paused Pol II complexes until Tat mediates the transfer of P-TEFb to TAR and subsequent kinase activation. Although the molecular details are not yet clear, there is evidence that Tat may utilize two aspects of molecular mimicry during the activation process: first, the Tat AD appears to compete with Hexim1 for a shared binding surface on CycT1, and second, the Tat RBD appears to recognize a TAR-like motif (GA-UC) within the 5= stem-loop of 7SK snRNA, as well as an additional feature in the 3=-end stem-loop (2,25,53,54,70,95). Thus, while Tat alone is able to displace Hexim1 and extract P-TEFb from the 7SK snRNP complex in vitro and in vivo (2,21,54), the Tat-TAR interaction likely also plays a significant role in the context of actively transcribing complexes in vivo.…”

mentioning

confidence: 99%

Transition Step during Assembly of HIV Tat:P-TEFb Transcription Complexes and Transfer to TAR RNA

D’Orso

Jang

Pastuszak

et al. 2012

Molecular and Cellular Biology

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bTranscription factors regulate eukaryotic RNA polymerase II (Pol II) activity by assembling and remodeling complexes at multiple steps in the transcription cycle. In HIV, we previously proposed a two-step model where the viral Tat protein first preassembles at the promoter with an inactive P-TEFb:7SK snRNP complex and later transfers P-TEFb to TAR on the nascent transcript, displacing the inhibitory snRNP and resulting in Pol II phosphorylation and stimulation of elongation. It is unknown how the Tat:P-TEFb complex transitions to TAR to activate the P-TEFb kinase. Here, we show that P-TEFb artificially recruited to the nascent transcript is not competent for transcription but rather remains inactive due to its assembly with the 7SK snRNP. Tat supplied in trans is able to displace the kinase inhibitor Hexim1 from the snRNP and activate P-TEFb, thereby uncoupling Tat requirements for kinase activation and TAR binding. By combining comprehensive mutagenesis of Tat with multiple cellbased reporter assays that probe the activity of Tat in different arrangements, we genetically defined a transition step in which preassembled Tat:P-TEFb complexes switch to TAR. We propose that a conserved network of residues in Tat has evolved to control this transition and thereby switch the host elongation machinery to viral transcription.

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Preformed Protein-binding Motifs in 7SK snRNA: Structural and Thermodynamic Comparisons with Retroviral TAR

Cited by 28 publications

References 40 publications

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

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

hLARP7 C-terminal domain contains an xRRM that binds the 3′ hairpin of 7SK RNA

Transition Step during Assembly of HIV Tat:P-TEFb Transcription Complexes and Transfer to TAR RNA

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