Resumen de Gemin5, a multifunctional RNA-binding protein involved in translation control.
RNA-binding proteins (RBPs) regulate all steps of RNA metabolism including protein synthesis, which is an essential stage of gene expression. Gemin5 is an RBP involved in the assembly of splicing machinery, gene expression reprogramming, and translation control. This protein is organized in domains that interact with distinct cellular targets. The N-terminal region comprises 14WD repeat motifs (residues 1-739) that recognize small nuclear RNAs, and bind to the ribosome, while the C-terminal end bears a bipartite RNA-binding site, designated as RBS1 and RBS2 (residues 1287-1508). RBS1 is a non-canonical RNA-binding domain (RBD) that recognizes a sequence located within the coding region of Gemin5 mRNA, termed H12.
Remarkably, Gemin5 stimulates its own translation through direct interaction between RBS1 and H12, providing a regulatory feedback loop that allows to fine-tune its cellular levels.
In this thesis, we have conducted an RNA-protein coevolution study that predicted the coevolving pairs between H12 and RBS1. The H12 RNA shows a stable secondary structure consisting of two stem-loops, SL1 and SL2. SL1 contains all coevolving nucleotides. Analysis of the RNAprotein interactions in vitro confirmed that SL1 is the main motif recognized by RBS1 in H12.
Biochemical and functional assays revealed that the PXSS motif in RBS1 is involved in the recognition of H12 through SL1. This study paves the way for the recognition of non-canonical RBDs carrying similar motifs.
In addition, we have solved the structure of a tetratricopeptide (TPR)-like domain in the middle region of Gemin5 (residues 845-1097) that self-assembles into a compact canoe-shaped dimer and acts as a protein-protein interaction platform. Despite the tight association of this dimer, a single point mutation (A951E) at the closest intersubunit distance was sufficient to destabilize it.
We have proven that this domain drives the dimerization of Gemin5 in living cells. These data indicate that the recruitment of the endogenous Gemin5 by p85, a viral cleavage fragment that includes the TPR and the RBS domains, might prevent the role of Gemin5 in translation control.
The presence of distinct RBDs in Gemin5 facilitates the interaction with a wide variety of cellular mRNAs. In order to identify the mRNAs selectively translated by Gemin5, we performed a genome-wide analysis of polysome-associated mRNAs in Gemin5-depleted cells relative to control cells. Among the transcripts displaying enhanced association to polysomes, there are mRNAs encoding for ribosomal proteins, histones, mitochondrial ribosomal proteins, proteins of cytochrome P450, and Sm and like-Sm proteins. Remarkably, we have found that Gemin5 stimulates translation of the TOP mRNA family, which includes all ribosomal protein transcripts.
Finally, the relevance of RNA structure for RNA-protein interactions prompted us to explore a new method based on tRNA scaffold to identify RBPs associated to RNA structural elements.
This novel approach will allow the identification of RBPs critical in different steps of RNA metabolism
