RESUMO
Mtr4 is an essential RNA helicase involved in nuclear RNA processing and degradation and is a member of the Ski2-like helicase family. Ski2-like helicases share a common core architecture that includes two RecA-like domains, a winged helix, and a helical bundle (HB) domain. In Mtr4, a short C-terminal tail immediately follows the HB domain and is positioned at the interface of the RecA-like domains. The tail ends with a SLYΦ sequence motif that is highly conserved in a subset of Ski2-like helicases. Here, we show that this sequence is critical for Mtr4 function. Mutations in the C-terminus result in decreased RNA unwinding activity. Mtr4 is a key activator of the RNA exosome complex, and mutations in the SLYΦ motif produce a slow growth phenotype when combined with a partial exosome defect in S. cerevisiae, suggesting an important role of the C-terminus of Mtr4 and the RNA exosome. We further demonstrate that C-terminal mutations impair RNA degradation activity by the major RNA exosome nuclease Rrp44 in vitro. These data demonstrate a role for the Mtr4 C-terminus in regulating helicase activity and coordinating Mtr4-exosome interactions.
Assuntos
Exossomos , Proteínas de Saccharomyces cerevisiae , Exossomos/genética , Exossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/química , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA Helicases DEAD-box/química , RNA Helicases/química , DNA Helicases/metabolismoRESUMO
The Ski2-like RNA helicase, Mtr4, plays a central role in nuclear RNA surveillance pathways by delivering targeted substrates to the RNA exosome for processing or degradation. RNA target selection is accomplished by a variety of Mtr4-mediated protein complexes. In S. cerevisiae, the Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex prepares substrates for exosomal decay through the combined action of polyadenylation and helicase activities. Biophysical and structural studies of Mtr4 and TRAMP require highly purified protein components. Here, we describe robust protocols for obtaining large quantities of pure, active Mtr4 and Trf4-Air2 from S. cerevisiae. The proteins are recombinantly expressed in E. coli and purified using affinity, ion exchange, hydrophobic exchange and size exclusion chromatography. Care is taken to remove nuclease contamination during the prep. Assembly of TRAMP is achieved by combining individually purified Mtr4 and Trf4-Air2. We further describe a strand displacement assay to characterize Mtr4 helicase unwinding activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Escherichia coli/genética , Escherichia coli/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA Helicases/química , RNA Helicases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
In this issue of Structure, Absmeier et al. (2020) describe the molecular mechanisms employed by an RNA helicase to prevent premature ATP hydrolysis upon nucleotide binding.