NMR structure of the three quasi RNA recognition motifs (qRRMs) of human hnRNP F and interaction studies with Bcl-x G-tract RNA: a novel mode of RNA recognition

The heterogeneous nuclear ribonucleoprotein (hnRNP) F belongs to the hnRNP H family involved in the regulation of alternative splicing and polyadenylation and specifically recognizes poly(G) sequences (G-tracts). In particular, hnRNP F binds a G-tract of the Bcl-x RNA and regulates its alternative splicing, leading to two isoforms, Bcl-xS and Bcl-xL, with antagonist functions. In order to gain insight into G-tract recognition by hnRNP H members, we initiated an NMR study of human hnRNP F. We present the solution structure of the three quasi RNA recognition motifs (qRRMs) of hnRNP F and identify the residues that are important for the interaction with the Bcl-x RNA by NMR chemical shift perturbation and mutagenesis experiments. The three qRRMs exhibit the canonical βαββαβ RRM fold but additional secondary structure elements are present in the two N-terminal qRRMs of hnRNP F. We show that qRRM1 and qRRM2 but not qRRM3 are responsible for G-tract recognition and that the residues of qRRM1 and qRRM2 involved in G-tract interaction are not on the β-sheet surface as observed for the classical RRM but are part of a short β-hairpin and two adjacent loops. These regions define a novel interaction surface for RNA recognition by RRM

NMR structure of the three quasi RNA recognition motifs (qRRMs) of human hnRNP F and interaction studies with Bcl-x G-tract RNA: a novel mode of RNA recognition

The heterogeneous nuclear ribonucleoprotein (hnRNP) F belongs to the hnRNP H family involved in the regulation of alternative splicing and polyadenylation and specifically recognizes poly(G) sequences (G-tracts). In particular, hnRNP F binds a G-tract of the Bcl-x RNA and regulates its alternative splicing, leading to two isoforms, Bcl-x(S) and Bcl-x(L), with antagonist functions. In order to gain insight into G-tract recognition by hnRNP H members, we initiated an NMR study of human hnRNP F. We present the solution structure of the three quasi RNA recognition motifs (qRRMs) of hnRNP F and identify the residues that are important for the interaction with the Bcl-x RNA by NMR chemical shift perturbation and mutagenesis experiments. The three qRRMs exhibit the canonical βαββαβ RRM fold but additional secondary structure elements are present in the two N-terminal qRRMs of hnRNP F. We show that qRRM1 and qRRM2 but not qRRM3 are responsible for G-tract recognition and that the residues of qRRM1 and qRRM2 involved in G-tract interaction are not on the β-sheet surface as observed for the classical RRM but are part of a short β-hairpin and two adjacent loops. These regions define a novel interaction surface for RNA recognition by RRMs

NMR-based docking of protein-protein complexes : the human UbcH5b-CNOT4 ubiquitination complex

Understanding the molecular and functional interactions among macromolecular complexes, as well as their changes associated with time, cell type or disease state will be invaluable for human health, and will have direct implications, for example, in pharmaceutical research to identify and select potential targets, and design efficient and specific drugs. Structural studies of macromolecular complexes, however, suffer from some limitations, especially in the case of weak and transient complexes. New and complementary methodologies, such as docking, have therefore been developed. The current computational approaches, however, also suffer from limitations and new developments and improvements are needed. This thesis introduces a new docking approach in which the docking of two macromolecules is driven by biophysical and/or biochemical information and furthermore describes structural studies on the UbcH5B-CNOT4 complex involved in the ubiquitination pathway. HADDOCK is a new docking approach that is based on biophysical and/or biochemical information. This information, derived for example from NMR chemical shift perturbation or site-directed mutagenesis experiments, is converted into highly ambiguous intermolecular distance restraints that are directly used to drive the docking process. The docking protocol allows for side chains and backbone flexibility at the interface and the solutions are scored according to an intermolecular energy term. The method was successfully tested on three complexes. The solution NMR structure of UbcH5B, an E2 ubiquitin conjugating enzyme has been solved. NMR relaxation measurements are performed on UbcH5B. They show limited motions for the major part of the protein backbone. We compare the structure of UbcH5B with other E2 structures, and the global fold of all E2s is very similar. Some differences are, however, observed and correlate well with the dynamical properties of E2s. The position and orientation of the N-terminal a-helix as compared to the core of the protein differ in the various structures. This difference may be determinant in E3 ubiquitin ligase binding and recognition. Furthermore, a highly conserved asparagine residue was shown to be important for the ubiquitin transfer. In crystal structures, this asparagine points away from the active site cysteine. Structure of UbcH5B shows that in solution, this asparagine is in close proximity to the active site cysteine, in a conformation suitable for its catalytic role. HADDOCK is then used to generate a structural model of the UbcH5B-CNOT4 complex. CNOT4 is an E3 ubiquitin ligase that is part of the CCR4-NOT complex involved in transcription repression. The residues of the CNOT4 RING domain important for the interaction with UbcH5B were previously reported. Here, the residues of UbcH5B important for the binding to CNOT4 RING are identified from NMR chemical shift perturbation experiments. These data are used to generate a structural model of the UbcH5B-CNOT4 complex. Two sets of solutions are, however, obtained that can not be discriminated. Mutagenesis experiments are performed and identify charged residues of UbcH5B (Lys63) and of CNOT4 (Glu49) involved in an electrostatic interaction. Once this information is included in the docking, a unique set of solutions is obtained. The structural model of the UbcH5B-CNOT4 complex is compared with the X-ray structure of the homologous UbcH7-c-Cbl complex and significant differences at specific residues give structural insights into the mechanisms of the E2-E3 specificity

The high kinetic stability of a G-quadruplex limits hnRNP F qRRM3 binding to G-tract RNA

The RNA binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) F is involved in telomeres maintenance and pre-mRNA processing, such as alternative splicing and polyadenylation. It specifically recognizes RNA containing three consecutive guanines (G-tracts) that have the potential to assemble into G-quadruplexes. We have proposed recently that hnRNP F could regulate alternative splicing by remodeling RNA structures, such as G-quadruplexes. However, the exact mechanism of hnRNP F binding to such RNA sequences remains unknown. Here, we have studied the binding of the third RNA binding domain of hnRNP F [quasi-RNA recognition motif 3 (qRRM3)] to G-tract RNA using isothermal titration calorimetry, circular dichroism and nuclear magnetic resonance spectroscopy. Our results show that qRRM3 binds specifically exclusively to single-stranded G-tracts (ssRNA), in contrast to previous reports stating that the G-quadruplex was recognized as well. Furthermore, we demonstrate that the pre-existent ssRNA/G-quadruplex equilibrium slows down the formation of the protein-ssRNA complex. Based on in vitro transcription assays, we show that the rate of the protein-RNA complex formation is faster than that of the G-quadruplex. We propose a model according to which hnRNP F could bind RNA co-transcriptionally and prevents G-quadruplex formatio

NR4A Nuclear Receptors Target Poly-ADP-Ribosylated DNA-PKcs Protein to Promote DNA Repair.

Although poly-ADP-ribosylation (PARylation) of DNA repair factors had been well documented, its role in the repair of DNA double-strand breaks (DSBs) is poorly understood. NR4A nuclear orphan receptors were previously linked to DSB repair; however, their function in the process remains elusive. Classically, NR4As function as transcription factors using a specialized tandem zinc-finger DNA-binding domain (DBD) for target gene induction. Here, we show that NR4A DBD is bi-functional and can bind poly-ADP-ribose (PAR) through a pocket localized in the second zinc finger. Separation-of-function mutants demonstrate that NR4A PAR binding, while dispensable for transcriptional activity, facilitates repair of radiation-induced DNA double-strand breaks in G1. Moreover, we define DNA-PKcs protein as a prominent target of ionizing radiation-induced PARylation. Mechanistically, NR4As function by directly targeting poly-ADP-ribosylated DNA-PKcs to facilitate its autophosphorylation-promoting DNA-PK kinase assembly at DNA lesions. Selective targeting of the PAR-binding pocket of NR4A presents an opportunity for cancer therapy

Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68

Sam68 and T-STAR are members of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins. T-STAR is a tissue-specific paralogue that regulates the alternative splicing of neuronal pre-mRNAs. STAR proteins differ from most splicing factors, in that they contain a single RNA-binding domain. Their specificity of RNA recognition is thought to arise from their property to homodimerize, but how dimerization influences their function remains unknown. Here, we establish at atomic resolution how T-STAR and Sam68 bind to RNA, revealing an unexpected mode of dimerization different from other members of the STAR family. We further demonstrate that this unique dimerization interface is crucial for their biological activity in splicing regulation, and suggest that the increased RNA affinity through dimer formation is a crucial parameter enabling these proteins to select their functional targets within the transcriptome

Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions

Sam68, also known as KHDRBS1, is a member of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins and its role is modulated by post-translational modifications, including serine/threonine phosphorylation, that differ at various stages of the cell cycle. However, the molecular basis and mechanisms of these modulations remain largely unknown. Here, we combined mass spectrometry, nuclear magnetic resonance spectroscopy and cell biology techniques to provide a comprehensive post-translational modification mapping of Sam68 at different stages of the cell cycle in HEK293 and HCT116 cells. We established that Sam68 is specifically phosphorylated at T33 and T317 by Cdk1, and demonstrated that these phosphorylation events reduce the binding of Sam68 to RNA, control its cellular localization and reduce its alternative splicing activity, leading to a reduction in the induction of apoptosis and an increase in the proliferation of HCT116 cells

2′-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions

Protein-RNA interactions are central to numerous cellular processes. In this work, we present an easy and straightforward NMR-based approach to determine the RNA binding site of RNA binding proteins and to evaluate the binding of pairs of proteins to a single-stranded RNA (ssRNA) under physiological conditions, in this case in nuclear extracts. By incorporation of a 19F atom on the ribose of different nucleotides along the ssRNA sequence, we show that, upon addition of an RNA binding protein, the intensity of the 19F NMR signal changes when the 19F atom is located near the protein binding site. Furthermore, we show that the addition of pairs of proteins to a ssRNA containing two 19F atoms at two different locations informs on their concurrent binding or competition. We demonstrate that such studies can be done in a nuclear extract that mimics the physiological environment in which these protein-ssRNA interactions occur. Finally, we demonstrate that a trifluoromethoxy group (-OCF3) incorporated in the 2′ribose position of ssRNA sequences increases the sensitivity of the NMR signal, leading to decreased measurement times, and reduces the issue of RNA degradation in cellular extracts