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

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.

Figure 1

Schematic of screen for RNA substrates of Ire1p endoribonuclease using an in vitro nuclease reaction. Recombinant Ire1* expressed and purified from E. coli is incubated with poly(A)⁺ RNA isolated from wild-type S. cerevisiae to cleave endogenous HAC1 mRNA and other potential RNA substrates of Ire1p. Cleaved RNA (lacking the poly(A) tail) is separated from uncleaved RNA as the unbound, poly(A)^- fraction from an oligo(dT) column and used to prepare fluorescent probe by reverse transcription followed by PCR amplification in the presence of Cy3-dTTP. A second control probe, using poly(A)^- RNA from mock nuclease reactions (identical reactions except for the lack of Ire1*) is prepared similarly, except that PCR amplification was carried out in the presence of Cy5-dTTP. Equal amounts of these probes are mixed and used to probe the yeast DNA microarray. Because RNA fragments generated by Ire1* cleavage are represented only in the Cy3 probe, microarray spots hybridizing to cleaved fragments should appear green, whereas microarray spots hybridizing to molecules common to both probes should appear yellow upon superimposition of green (Cy3) and red (Cy5) channels.

Figure 2

Efficient cleavage of HAC1 mRNA by Ire1* in the presence of cellular mRNA. (a) Schematic diagram of the Ire1*-cleaved (poly(A)^-) and uncleaved (poly(A)⁺) RNA fractions separated after an in vitro nuclease reaction on yeast poly(A)⁺ RNA. (b) Northern blot of the RNA fractions indicated in (a) probed with a PCR fragment encompassing either the 5' exon of HAC1 (lanes 1-3) or the 3' exon (lanes 4-6). Lanes 1 and 4, yeast poly(A)⁺ RNA before Ire1* cleavage; lanes 2 and 5, bound uncleaved RNA fraction (b, poly(A)⁺); lanes 3 and 6, unbound cleaved RNA fraction (u, poly(A)^-). Positions of uncleaved and cleaved HAC1 mRNA are indicated. Note that the 5' exon and 5' exon plus intron, and the 3' exon and 3' exon plus intron RNA species, respectively, co-migrate on these agarose gels.

Figure 3

HAC1 is a unique RNA cleaved by Ire1*. (a) Scatter plot of Cy3 and Cy5 signal intensities following hybridization to a yeast ORF microarray with both Cy3- and Cy5-labeled probes, prepared from the cleaved RNA fragments generated from either Ire1* or mock treatment, respectively, as described in Figure 1. Each point on the plot represents a single yeast ORF. Points below the diagonal represent ORFs that hybridize predominantly to RNAs represented in the Cy3 probe. The position of the spot displaying the brightest Cy3 signals corresponds to HAC1. (b) Histogram representation of the log₂ Cy3/Cy5 ratio. Inset, HAC1 is the only gene with a log₂ Cy3/Cy5 ratio near 2.3. All data displayed in Figure 3 are provided in Additional data file 1.

Figure 4

HAC1 stem-loop motif used for the genome wide computational consensus search. (a) Predicted secondary structure and sequence flanking both 5' and 3' Ire1p cleavage sites in HAC1 mRNA. Cleavage occurs after a G residue located at the third position of the seven-nucleotide loop. (b) Parameters used for the computer search. A four base-pair stem with a seven-nucleotide loop with C at the first position, and G both at third and sixth positions, are determined experimentally as described previously [14]. Possible base-pairs (N = N') in the stem include AU, UA, GC, CG, UG, GU. The complete output of the computational screen is listed in Additional data file 2.

Figure 5

HAC1 is the only transcript decreasing in rlg1-100 cells following tunicamycin treatment. (a) Scatter plot of Cy3 and Cy5 signal intensities following hybridization to a yeast ORF microarray with both Cy3- and Cy5-labeled probes, prepared from RNA isolated from rlg1-100 cells either untreated or treated with tunicamycin for 40 min, respectively. Points below the diagonal represent ORFs that hybridize predominantly to RNAs represented in the Cy3 probe, and which are therefore present at reduced levels upon tunicamycin treatment. The position of the spot displaying the brightest Cy3 signals (corresponding to the HAC1) is shown. (b) Histogram presentation of microarray data measuring log₂ of the relative mRNA abundance between tunicamycin treated (40 min) and untreated rlg1-100 cells. Levels of most mRNAs were not altered, and the only mRNA significantly changed upon tunicamycin treatment is HAC1. Inset, HAC1 is the only gene with a log₂ fold-change in abundance near 1.5. All data displayed in Figure 5 are provided in Additional data file 3.