Combined Nuclear Magnetic Resonance and spin labeling approach for the structural and dynamic studies of RNA/proteins complexes involved in processes of regulation: Application to 7SK RNA, regulator of the transcription by RNA polymerase II.
RNAs and their complexes (RNP) are involved in many steps of biological processes. The elucidation of mechanisms of interactions between RNAs and their partners is now a major objective in molecular and structural biology. The key to understand the biological functions of such complexes is to investigate their structure, their plasticity and how these complexes evolve with time. The Nuclear Magnetic Resonance (NMR) is a powerful technique to investigate the structural and dynamical properties of RNAs. However, the study of large RNAs by NMR remains challenging, due the combined effect of efficient relaxation and spectral crowding. At the same time, the Electronic Paramagnetic Resonance (EPR) has rapidly expanded to study the structure and the dynamic of macromolecules. <br />The “RNASPIN” project aims at developing an innovative approach for the study of RNP complexes using NMR combined to EPR. In particular, we finalize new techniques based on the incorporation of paramagnetic probes at specific position in the RNA, coupled to the measurement of the induced effects on nuclear spins. <br />At the term of the project, protocols of labeling, structural studies and modeling program will be available to be applied to others complexes involved in regulatory processes such as the 7SK RNA, involved in the regulation of the transcription by RNA polymerase II.
In a recent work, we studied the interaction between the 5’-terminal hairpin of 7SK and the NLS domain of the protein Hexim1 using NMR. We identified several crucial elements in 7SK RNA for Hexim1 recognition (Lebars et al., 2010). At that time, no structural and dynamical data at molecular level are available concerning the structure of the free 7SK RNA and interaction with its partners.
Our project aims at solving the solution structure of the 5’ hairpin of the 7SK RNA using NMR to gain insight into the basis of the recognition mechanism between this RNA and the NLS domain of Hexim1.
The project is divided in four major tasks:
(i) The production of samples compatible to NMR studies;
(ii) The implementation and the development of the spin labeling techniques for the combined NMR/EPR studies of RNAs and their complexes;
(iii) The NMR data experiments and their analysis followed by molecular modeling to solve the solution structure of the RNA ;
(iv) Application of the combined NMR/EPR approach to the 7SK RNP (analysis of information derived from experiments with the spin label RNA; incorporation of the data in the molecular modeling protocol).
We provide a fully enzymatic method which allows the insertion of a paramagnetic center at specific position(s) in an RNA molecule. Our Method enables various combinations of labeling schemes, a strategy of interest for NMR and EPR studies.
The paramagnetic approach that we are currently developing will be applied to refine the 3D structure of an RNA that we solved using NMR and to study the interaction of this RNA with its partners.
At the term of the project, protocols of labeling, of structural studies and modeling program will be applied to more complexes systems involved in regulation processes normally not accessible by NMR.
The method that we provide has been published in an international journal with a reviewing process:
I. Lebars*, B. Vileno, S. Bourbigot, P. Turek, P. Wolff & B. Kieffer, 2014, «A fully enzymatic method for site-directed spin-labeling of long RNA«, Nucleic Acids Res., 42(15):e117, doi: 10.1093/nar/gku553.
The results have not yet been published.
RNAs play a crucial role in all steps of gene expression. As intermediate between DNA and proteins, RNAs are the carriers of the genetic code and of informations that allow the regulation of translation, depending on their sequences and their ability to fold into secondary and tertiary structures. RNA molecules folded into complex structures are involved in many biological processes in all organisms. Such structures are stabilized by hydrogen bonds networks, stacking and electrostatic interactions.
RNAs structures also provide binding site for ligands, proteins or nucleic acids. RNA/protein complexes are effectively involved in many steps of cellular function, genetic or microbial infection. Modulation of RNA-protein recognition events involved in transcription, mRNA processing, translation or development of retroviruses could lead to the conception of new therapeutic agents.
The elucidation of mechanisms of interactions between RNAs and their partners is now a major objective in molecular and structural biology. Studies of RNA-protein recognition are critical to understand how gene expression is regulated. The key to understand the biological functions of such complexes is to investigate their structure and how these complexes evolve in time (dynamical properties). Nuclear Magnetic Resonance (NMR) is the most powerful tool for studying the dynamic of RNAs and their interactions with partners in solution. In addition, over the recent years, several techniques have been developed to overcome the limits of NMR. At the same time, Electronic Paramagnetic Resonance (EPR) has rapidly expanded over the last years to study structure and dynamics of macromolecules and complexes. For a long time, this technique was applied successfully to proteins. Only recently, EPR was also used to characterize interactions between RNAs and proteins.
In the project presented here, we propose to initiate and to develop a combined NMR and EPR approach for the structural and dynamic study of RNA/protein subunit of the 7SK/HEXIM1/P-TEFb/LaRP7/MePCE ribonucleoprotein complex involved in the regulation of the development of several pathologies as cancer, inflammation, AIDS and cardiac hypertrophy.
The P-TEFb complex (« positive transcription elongation factor b ») formed by the kinase cyclin-dependent Cdk9 and the cyclin T1/T2, activates transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNApol II). This complex is an essential regulator of gene expression. The 7SK RNA, discovered as an abundant snRNA in the middle seventies, acts as a regulator of transcription by RNApol II, in cooperation with the protein HEXIM1 (or the minor HEXIM2 protein), by sequestering P-TEFb into the ribonucleoprotein complex 7SK/HEXIM-1/P-TEFb.
Recent work has revealed that P-TEFb and HEXIM1 are connected to other partners: the La-related protein LaRP7 and the methylphosphate capping enzyme MePCE. LaRP7 modulates transcription by binding the conserved 3’ UUU-OH region of 7SK and by the sequestration of the P-TEFb complex. For the activation of the transcription, P-TEFb is released and LaRP7 still binds the RNA. MePCE that contains a methyltransferase domain, adds one monomethyl phosphate cap structure onto the 5’of 7SK. LaRP7 and MePCE act cooperatively to ensure the stability of 7SK and to promote the 7SK RNP assembly. Actually, no structural and dynamic data at molecular level are available concerning the structure of the RNA 7SK in interaction with its partners.
Our project is to characterize interactions between 7SK hairpin 1 and HEXIM1, on one hand, and 7SK hairpin 4 and LaRP7, on the other hand, using NMR end EPR. Structural data will allow the elucidation of the mechanism of recognition between hairpins of 7SK and these proteins. This study will bring additional information to elucidate the major role of 7SK in the regulation of transcription by RNApol II.
Madame Isabelle LEBARS (CENTRE EUROPEEN DE RECHERCHE EN BIOLOGIE ET EN MEDECINE - CERBM) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IGBMC CENTRE EUROPEEN DE RECHERCHE EN BIOLOGIE ET EN MEDECINE - CERBM
Help of the ANR 300,000 euros
Beginning and duration of the scientific project: - 36 Months