Genome-scale approaches for discovering novel nonconventional splicing substrates of the Ire1 nuclease

Genome Biol. 2005;6(1):R3. doi: 10.1186/gb-2004-6-1-r3. Epub 2004 Dec 22.

Abstract

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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alternative Splicing / genetics*
  • Basic-Leucine Zipper Transcription Factors
  • Computational Biology*
  • Consensus Sequence / genetics
  • Genome, Fungal*
  • Genomics / methods*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Oligonucleotide Array Sequence Analysis
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • RNA Ligase (ATP) / metabolism
  • RNA, Fungal / genetics
  • RNA, Fungal / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Repressor Proteins / genetics
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sensitivity and Specificity
  • Substrate Specificity
  • Transcription Factors / genetics

Substances

  • Basic-Leucine Zipper Transcription Factors
  • HAC1 protein, S cerevisiae
  • Membrane Glycoproteins
  • RNA, Fungal
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • IRE1 protein, S cerevisiae
  • Protein-Serine-Threonine Kinases
  • RNA Ligase (ATP)