Protomer alignment modulates specificity of RNA substrate recognition by Ire1

Area of Science:

• Molecular Biology
• Cellular Biology
• Biochemistry

Background:

• The unfolded protein response (UPR) is crucial for maintaining protein folding homeostasis in the endoplasmic reticulum (ER).
• The Ire1 branch of the UPR in metazoan cells has two outputs: mRNA splicing and selective mRNA decay (RIDD).
• Ire1 orthologs in yeast species are specialized for either splicing (Saccharomyces cerevisiae) or RIDD (Schizosaccharomyces pombe).

Purpose of the Study:

• To investigate the molecular basis for the functional specialization of Ire1's RNase activity.
• To develop an assay for measuring Ire1 RNase promiscuity.
• To understand how Ire1 protomer alignment influences substrate specificity.

Main Methods:

• Development of a novel assay to report on Ire1 RNase promiscuity.
• Site-directed mutagenesis to convert amino acids in Saccharomyces cerevisiae Ire1 to Schizosaccharomyces pombe counterparts.
• Biochemical assays and computational modeling to analyze Ire1 RNase domain structure and function.

Main Results:

• Conversion of two amino acids in Saccharomyces cerevisiae Ire1's RNase domain conferred promiscuous activity, similar to Schizosaccharomyces pombe Ire1.
• These mutations were shown to rewire salt bridges at the Ire1 RNase domain's dimer interface.
• The altered salt bridges resulted in changed protomer alignment within the Ire1 dimer.

Conclusions:

• Ire1 protomer alignment is a critical determinant of its RNase substrate specificity.
• The study reveals a mechanism for functional specialization within the UPR pathway.
• Understanding Ire1's RNase activity regulation offers insights into protein homeostasis.