Structural basis of Nrd1-Nab3 heterodimerization.

Belén Chaves-Arquero,Olga Calvo,Ramón Campos-Olivas,Sergio Camero,Yasmina Mirassou,José Manuel Pérez-Cañadillas,Clara M Santiveri,Santiago Martínez-Lumbreras,Maria Ángeles Jiménez

doi:10.26508/lsa.202101252

Abstract

Heterodimerization of RNA binding proteins Nrd1 and Nab3 is essential to communicate the RNA recognition in the nascent transcript with the Nrd1 recognition of the Ser5-phosphorylated Rbp1 C-terminal domain in RNA polymerase II. The structure of a Nrd1-Nab3 chimera reveals the basis of heterodimerization, filling a missing gap in knowledge of this system. The free form of the Nrd1 interaction domain of Nab3 (NRID) forms a multi-state three-helix bundle that is clamped in a single conformation upon complex formation with the Nab3 interaction domain of Nrd1 (NAID). The latter domain forms two long helices that wrap around NRID, resulting in an extensive protein-protein interface that would explain the highly favorable free energy of heterodimerization. Mutagenesis of some conserved hydrophobic residues involved in the heterodimerization leads to temperature-sensitive phenotypes, revealing the importance of this interaction in yeast cell fitness. The Nrd1-Nab3 structure resembles the previously reported Rna14/Rna15 heterodimer structure, which is part of the poly(A)-dependent termination pathway, suggesting that both machineries use similar structural solutions despite they share little sequence homology and are potentially evolutionary divergent.

Highlights

The missing cross-peaks could be explained by conformational exchange broadening and/or participation of those regions in high molecular weight oligomerization, whose broad nuclear magnetic resonance (NMR) line widths are beyond detection, leaving the flexible tails with faster dynamics as the only “visible” parts in NMR
The circular dichroism (CD) spectrum of Nrd1 NAID (Fig 1D in blue) reveals a mixture of unstructured and α-helical conformation, which points to the α-helical nature of these hypothetical oligomers
The reported structure of the Nrd1–Nab3 chimera allows studying the relevance of Nab3/Nrd1 heterodimerization in vivo, by designing mutations that potentially destabilize this interaction, as we did for the Nrd1 RNA-binding domain (RBD) (Franco-Echevarrıa et al, 2017)

Summary

Introduction

The mechanisms of transcription termination have been profusely studied from different approaches; from cell biology to structural methods (Richardson, 1996; Birse et al, 1998; Dichtl & Keller, 2001; Mischo & Proudfoot, 2013; Arndt & Reines, 2015; Lemay & Bachand, 2015; Porrua & Libri, 2015). In the Saccharomyces cerevisiae model system there, are two different transcription termination mechanisms: the poly(A)-dependent pathway that mainly processes mRNAs (Birse et al, 1998; Dichtl & Keller, 2001) and the poly(A)independent pathway that processes most of the short noncoding transcripts such as snoRNAs (Conrad et al, 2000; Carroll et al, 2004, 2007; Kim et al, 2006) This latter pathway principally involves three proteins, Nrd, Nab, and Sen, and is referred to as the Nrd1Nab3-Sen (NNS) pathway.

Methods

Results

Conclusion