Sen1 has unique structural features grafted on the architecture of the Upf1-like helicase family.

Bronislava Leonaitė,Domenico Libri,Odil Porrua,Jérôme Basquin,Elena Conti,Fabien Bonneau,Zhong Han

doi:10.15252/embj.201696174

Abstract

The superfamily 1B (SF1B) helicase Sen1 is an essential protein that plays a key role in the termination of non‐coding transcription in yeast. Here, we identified the ~90 kDa helicase core of Saccharomyces cerevisiae Sen1 as sufficient for transcription termination in vitro and determined the corresponding structure at 1.8 Å resolution. In addition to the catalytic and auxiliary subdomains characteristic of the SF1B family, Sen1 has a distinct and evolutionarily conserved structural feature that “braces” the helicase core. Comparative structural analyses indicate that the “brace” is essential in shaping a favorable conformation for RNA binding and unwinding. We also show that subdomain 1C (the “prong”) is an essential element for 5′‐3′ unwinding and for Sen1‐mediated transcription termination in vitro. Finally, yeast Sen1 mutant proteins mimicking the disease forms of the human orthologue, senataxin, show lower capacity of RNA unwinding and impairment of transcription termination in vitro. The combined biochemical and structural data thus provide a molecular model for the specificity of Sen1 in transcription termination and more generally for the unwinding mechanism of 5′‐3′ helicases.

Highlights

In yeast, there are two major transcription termination pathways
To obtain a soluble fragment of Sen1 encompassing the helicase core, we subcloned a fragment of Sen1 cDNA coding for residues 976–1,904 into a vector for bacterial expression
Common to all Upf1-like helicases is the ability to unwind nucleic acids in the 50-30 direction. We propose that this property is based on a common molecular mechanism of unwinding that depends on the presence of similar structural elements

Summary

Introduction

There are two major transcription termination pathways. In the case of canonical protein-coding genes, Pol II normally terminates transcription via the cleavage and polyadenylation factor (CPF) complex, yielding stable mature mRNAs that are exported to the cytoplasm (reviewed in Mischo & Proudfoot, 2013). In the case of non-coding RNAs, such as cryptic unstable transcripts (CUTs) and small nucleolar RNAs (snoRNAs), Pol II terminates transcription via a non-canonical pathway that is coupled to RNA degradation (reviewed in Jensen et al, 2013). This non-canonical termination pathway depends on the Nrd1-Nab3-Sen (NNS) complex (reviewed in Arndt & Reines, 2015; Porrua & Libri, 2015a). While Nrd1-Nab couples the NNS complex to RNA degradation, Sen is the key enzyme in the transcription termination reaction (Porrua & Libri, 2013). Sen has been shown to be involved in termination of short protein-coding genes (Steinmetz et al, 2006), and inactivation of Sen leads to the accumulation of R-loops (RNA:DNA hybrids that form during transcription when the nascent RNA invades the DNA template) (Mischo et al, 2011)

Methods

Results

Conclusion