Structural identification of TERRA under physiological conditions and stress response

The recent identification of the TERRA locus carried out by our group opens the possibility of understanding the in vivo role of TERRA, something that is still unknown. In mice, the main locus is located on chromosome 18, while in humans it is located on chromosome 20q, knowing the transcripts produce at these loci Chr18-TERRA and Chr20q-TERRA, respectively. Using KO cells for Chr20q-TERRA allowed us to demonstrate that TERRAs are essential for telomeric maintenance and protection. However, it is known very little about the TERRA structure and how it determines its interaction with the telomere and its associated proteins.

For this purpose, several milestones are proposed that will be addressed in collaboration with other groups of the RyPSE consortium:

•  Milestone 1. Determine the structure of TERRA after the induction of stress.
•  Milestone 2. Determine the proteins that bind to TERRA after stress and see if their binding e changes TERRA structure.
•  Milestone 3. Role of nuclear bodies in the structure of TERRA and its response to stress.
•  Milestone 4. Study of the antitumoral role of ruthenium complexes in the murine model of cancer induced by c-myc and if this potential effect it is mediated by TERRA.

Functional and structural analysis of Gemin5 interacting RNAs and proteins

Regulation of proteins synthesis is a homeostasis control that allows a fast response to intracellular and environmental signals. Various versatile RNA-binding proteins (RBPs) are involved in this process, selectively modulating translation of specific mRNAs. Gemin5 controls translation through ribosome interaction, as well as binding to IRES elements and a region of its own mRNA (Francisco-Velilla et al, 2018). This feature relies on separate protein domains WD40, RBS1 and RBS2 (Figure 1). In this project we propose to decipher the three-dimensional structure of the RBS1 domain, as well as to determine the secondary structure of RNAs interacting with Gemin5.

To achieve this objective we propose specific aims in collaboration with members of the RyPSE consortium.

• Aim 1. Analysis of RNA reactivity towards dimetallic compounds (collaboration with UCM)
• Aim 2. Functional and structural characterization of the non-canonical RNA-binding motif of Gemin5 (collaboration with IQFR)
• Aim 3. Structural analysis of cellular RNAs interacting with Gemin5 (collaboration with UCM, CNIO y IQFR)
• Aim 4. Study of conformational changes in lncRNAs (collaboration with CNIO).

To study of the Intrinsically Disordered Domains of Gemin5 and hnRNP A1 and their interaction with nucleic acids and proteins

RNA binding proteins (RBPs) Gemin5 y hnRNP A1 display a typical domain organization made of folded RNA binding domains and intrinsically unstructured regions (IDD) (Figure 1). Both proteins interact with a large variety of RNAs through specific domains. In this project, we are interested in the interaction with viral RNA IRES and cellular lnc TERRAS, and the role that IDD regions play in this recognition process. Besides, hnRNP A1 interacts with structural elements for antiapoptotic genes which function as cellular IRES is still to be studied in detail.

To achieve this general objective, several milestones will be tackled in close collaboration with other groups of the RyPSE consortium.

• Aim  1. NMR studies of Intrinsically Disordered Domain of Gemin5 and hnRNP A1.
• Aim  2. To study interactions between Gemin5 IDD and domains of RNA IRES (in coordination with CBMSO group).
• Aim 3. NMR study of complexes between hnRNP A1 and lnc TERRA IRES (in coordination with CNIO and CBMSO groups).
• Aim  4. Interaction studies between hnRNP A1 and 5’-UTR structural elements of antiapoptotic genes IRES (in coordination with CBMSO and UCM groups).

Synthesis of diruthenium compounds for the structural characterization of biological species and to be tested as anticancer agents

One of the objectives of this project is preparing a library of diruthenium(II,III) compounds that can interact with proteins and different ARN and test their potential therapeutic use.

Thus, the following objectives are proposed in collaboration with the partner groups of the consortium:

• Objective 1. Synthesis and characterization of diruthenium compounds
• 1a. Open-paddlewheel compounds of the [Ru2Cl2(L-L)3] type: [Ru2Cl2(DPhF)3(DMSO)] (DPhF = N,N-difenilformamidinato) distinguish flexible regions of ARN (see Figure below). Similar compounds with different electronic and steric properties are aimed, in order to interact with different regions of the RNA.

Schematic representation of diruthenium paddlewheel (left) and open-paddlewheel (right) compounds. Reactive positions in the open-paddlewheel compound are marked with an asterisk. B. ORTEP diagram representation of the crystal structure of compound [Ru₂Cl₂(µ-DPhF)₃(DMSO)] (OPW-Ru). For clarity, only non-hydrogen atoms are shown. Lozano et al. RNA 22 (2016) 1-9.

• 1b. Paddlewheel compounds of the [Ru2Cl(O2CR)4] type: Compounds soluble in aqueous media and formed by ligands with demonstrated antitumor activity are aimed.

• Objective 2. Reactivity of diruthenium compounds with biological species
• 2a. Preparation of dirutenium-protein systems and characterization by NMR
• 2b. Study of the interactions of diruthenium compounds with oligonucleotides instead of RNA.