Genome-scale approaches for discovering novel nonconventional splicing substrates of the Ire1 nuclease.
BACKGROUND
The unfolded protein response (UPR) allows intracellular feedback regulation that adjusts the protein-folding capacity of the endoplasmic reticulum (ER) according to need. The signal from the ER lumen is transmitted by the ER-transmembrane kinase Ire1, which upon activation displays a site-specific endoribonuclease activity. Endonucleolytic cleavage of the intron from the HAC1 mRNA (encoding a UPR-specific transcription factor) is the first step in a nonconventional mRNA splicing pathway; the released exons are then joined by tRNA ligase. Because only the spliced mRNA is translated, splicing is the key regulatory step of the UPR.


RESULTS
We developed methods to search for additional mRNA substrates of Ire1p in three independent lines of genome-wide analysis. These methods exploited the well characterized enzymology and genetics of the UPR and the yeast genome sequence in conjunction with microarray-based detection. Each method successfully identified HAC1 mRNA as a substrate according to three criteria: HAC1 mRNA is selectively cleaved in vitro by Ire1; the HAC1 mRNA sequence contains two predicted Ire1 cleavage sites; and HAC1 mRNA is selectively degraded in tRNA ligase mutant cells.


CONCLUSIONS
Within the limits of detection, no other mRNA satisfies any of these criteria, suggesting that a unique nonconventional mRNA-processing mechanism has evolved solely for carrying out signal transduction between the ER and the nucleus. The approach described here, which combines biochemical and genetic 'fractionation' of mRNA with a novel application of cDNA microarrays, is generally applicable to the study of pathways in which RNA metabolism and alternative splicing have a regulatory role.
Niwa
Maho
M
Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94143-2200, USA. niwa@ucsd.edu
Patil
Christopher K
CK
DeRisi
Joe
J
Walter
Peter
P
D017398:Alternative Splicing / Q000235:genetics*; D050976:Basic-Leucine Zipper Transcription Factors; D019295:Computational Biology; D016384:Consensus Sequence / Q000235:genetics; D016681:Genome, Fungal; D023281:Genomics / Q000379:methods*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011130:RNA Ligase (ATP) / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics
D050976:Basic-Leucine Zipper Transcription Factors; C091341:HAC1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C082863:IRE1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D011130:RNA Ligase (ATP)