Abstract The conserved SR-like protein Npl3 promotes splicing of diverse pre-mRNAs. However, the RNA sequence(s) recognized by the RNA Recognition Motifs (RRM1 & RRM2) of Npl3 during the splicing reaction remain elusive. Here, we developed a split-iCRAC approach in yeast to uncover the consensus sequence bound to each RRM. High-resolution NMR structures show that RRM2 recognizes a 5´-GNGG-3´ motif leading to an unusual mille-feuille topology. These structures also reveal how RRM1 preferentially interacts with a CC-dinucleotide upstream of this motif, and how the inter-RRM linker and the region C-terminal to RRM2 contribute to cooperative RNA-binding. Structure-guided functional studies show that Npl3 genetically interacts with U2 snRNP specific factors and we provide evidence that Npl3 melts U2 snRNA stem-loop I, a prerequisite for U2/U6 duplex formation within the catalytic center of the B act spliceosomal complex. Thus, our findings suggest an unanticipated RNA chaperoning role for Npl3 during spliceosome active site formation.
U1 small nuclear ribonucleoparticle (U1 snRNP) plays a central role during RNA processing. Previous structures of U1 snRNP revealed how the ribonucleoparticle is organized and recognizes the pre-mRNA substrate at the exon-intron junction. As with many other ribonucleoparticles involved in RNA metabolism, U1 snRNP contains extensions made of low complexity sequences. Here, we developed a protocol to reconstitute U1 snRNP in vitro using mostly full-length components in order to perform liquid-state NMR spectroscopy. The accuracy of the reconstitution was validated by probing the shape and structure of the particle by SANS and cryo-EM. Using an NMR spectroscopy-based approach, we probed, for the first time, the U1 snRNP tails at atomic detail and our results confirm their high degree of flexibility. We also monitored the labile interaction between the splicing factor PTBP1 and U1 snRNP and validated the U1 snRNA stem loop 4 as a binding site for the splicing regulator on the ribonucleoparticle. Altogether, we developed a method to probe the intrinsically disordered regions of U1 snRNP and map the interactions controlling splicing regulation. This approach could be used to get insights into the molecular mechanisms of alternative splicing and screen for potential RNA therapeutics.
Abstract Ribonucleases (RNases) are ubiquitous enzymes that process or degrade RNA, essential for cellular functions and immune responses. The EndoU-like superfamily includes endoribonucleases conserved across bacteria, eukaryotes, and certain viruses, with an ancient evolutionary link to the ribonuclease A-like superfamily. Both bacterial EndoU and animal RNase A share a similar fold and function independently of cofactors. In contrast, the eukaryotic EndoU catalytic domain requires divalent metal ions for catalysis, possibly due to an N-terminal extension near the catalytic core. In this study, we used biophysical and computational techniques along with in vitro assays to investigate the calcium-dependent activation of human EndoU. We determined the crystal structure of EndoU bound to calcium and found that calcium binding remote from the catalytic triad triggers water-mediated intramolecular signaling and structural changes, activating the enzyme through allostery. Calcium-binding involves residues from both the catalytic core and the N-terminal extension, indicating that the N-terminal extension interacts with the catalytic core to modulate activity in response to calcium. Our findings suggest that similar mechanisms may be present across all eukaryotic EndoUs, highlighting a unique evolutionary adaptation that connects endoribonuclease activity to cellular signaling in eukaryotes.
Human THAP1 is the prototype of a large family of cellular factors sharing an original THAP zinc-finger motif responsible for DNA binding. Human THAP1 regulates endothelial cell proliferation and G1/S cell-cycle progression, through modulation of pRb/E2F cell-cycle target genes including rrm1. Recently, mutations in THAP1 have been found to cause DYT6 primary torsion dystonia, a human neurological disease. We report here the first 3D structure of the complex formed by the DNA-binding domain of THAP1 and its specific DNA target (THABS) found within the rrm1 target gene. The THAP zinc finger uses its double-stranded β-sheet to fill the DNA major groove and provides a unique combination of contacts from the β-sheet, the N-terminal tail and surrounding loops toward the five invariant base pairs of the THABS sequence. Our studies reveal unprecedented insights into the specific DNA recognition mechanisms within this large family of proteins controlling cell proliferation, cell cycle and pluripotency.
La famille des proteines THAP est caracterisee par la presence d'un motif proteique, le domaine THAP. Le domaine THAP de la proteine hTHAP1 defini un nouveau motif de coordination du zinc de type C2CH responsable de l'activite de liaison a l'ADN necessaire pour la fonction de facteur de transcription de la proteine hTHAP1, plus particulierement implique dans la regulation de la proliferation cellulaire. Le domaine THAP est caracterise sur le plan structural par un repliement atypique presentant une coordination tetraedrique du zinc et une longue insertion entre les deux paires de ligand du zinc adoptant un repliement de type sas. Le mode de reconnaissance specifique de l'ADN du domaine THAP a ete elucide par Resonance Magnetique Nucleaire. Ce domaine reconnait la cible ADN consensus 5'-TXXGGGCA-3' en etablissant des contacts bases specifiques par l'intermediaire de son extremite N-terminale, son brin s, la boucle L3 et la boucle L4. La resolution de structure du complexe THAP-ADN permet de comprendre comment le domaine THAP va reconnaitre specifiquement l'ADN, l'etape initiale permettant la regulation transcriptionnelle realisee par hTHAP1. Recemment, des mutations sur le gene de hTHAP1 ont ete genetiquement reliees a l'apparition d'une maladie neurodegenerative, la dystonie DYT6. Certaines de ces mutations perturbent la fonction du domaine THAP de hTHAP1 mettant ainsi en evidence que l'activite de liaison a l'ADN de hTHAP1 et la fonction de hTHAP1 sont cruciales pour le maintien de l'integrite des voies neuronales motrices.
