La-motif-dependent mRNA association with Slf1 promotes copper detoxification in yeast

Abstract

The La-motif (LAM) is an ancient and ubiquitous RNA-binding domain defining a superfamily of proteins, which comprises the genuine La proteins and La-related proteins (LARPs). In contrast to La, which binds and stabilizes pre-tRNAs and other RNA polymerase III transcripts, data on function and RNA targets of the LARPs have remained scarce. We have undertaken a global approach to elucidate the previously suggested role of the yeast LARP Slf1p in copper homeostasis. By applying RNA-binding protein immunopurification-microarray (RIP-Chip) analysis, we show that Slf1p and its paralog Sro9p copurify with overlapping sets of hundreds of functionally related mRNAs, including many transcripts coding for ribosomal proteins and histones. Interestingly, among these potential RNA targets were also mRNAs coding for proteins critical for protection of cells against elevated copper concentrations. Mutations introduced in the conserved aromatic patch of the LAM in Slf1p drastically impaired both association with its targets and Slf1-mediated protection of cells against toxic copper concentrations. Furthermore, we show that Slf1p stabilizes copper-related mRNA targets in a LAM-dependent manner. These results provide the first evidence for post-transcriptional regulation of factors/pathways implicated in copper homeostasis by a cytoplasmic RBP.

FIGURE 1.

Domain structure of selected LARP1 and La proteins and multiple-sequence alignment of LAMs. (A) Protein domain structure as defined by the National Center for Biotechnology Information Conserved domain finder (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cg). Motifs are depicted as boxes: black, LAM; light gray, RRM1; dark gray, RRM2; diagonally striped, RRM-L5; and vertically striped, DM15 motif. (B) Multiple sequence alignment of LAMs. The entire LAM of Slf1p and Sro9p comprised of 63 and 62 amino acids, respectively, is shown. Residues that are >80% conserved are boxed in gray. The identity and position of residues critical for terminal U binding in La proteins are shown on the top (numbering according to the Slf1 LAM). Asterisks highlight three aromatic amino acids that were changed to alanine in the Slf1 La-mutant. Species name abbreviations are as follows: S.c., Saccharomyces cerevisiae; C.e., Caenorhabditis elegans; D.m., Drosophila melanogaster; H.s., Homo sapiens; and T.b., Trypanosoma brucei.

FIGURE 2.

Copper resistance of SLF1 overexpressing cells depends on a functional LAM. (A) Yeast cells harboring a plasmid for gal-dependent expression of SLF1, SRO9, LHP1, or the empty vector were serially diluted (1:10) and spotted on SCgal-Ura plates containing indicated concentrations of CuSO₄. (B) Same assay comparing WT (pSLF1) and Slf1 La-mutant (pLAM-m) expressing cells. Pictures were taken after incubation of plates for 3–5 d at 30°C.

FIGURE 3.

Slf1p and Sro9p bind to overlapping sets of mRNAs. (A) Heat map depicting the log₂ ratios for RNA enrichment in three biological RIP replicates with Slf1p, Sro9p, and the negative control Fpr1p (columns). Rows are features representing transcripts and were grouped into Slf1-specific (211 transcripts), Sro9 and Slf1 associated transcripts (339), and Sro9-specific targets (501 transcripts). Each target group is ranked according to increasing P-values. The color code indicates the degree of enrichment (green–red color scale). mRNAs related to copper-homeostasis (red) and/or response to oxidative stress (black) are indicated to the right (for complete list, see Supplemental Table 2). (B) Venn-diagram depicting the overlap between Slf1p and Sro9p mRNA targets. The P-value was calculated with the Fisher's exact test. (C) Bar chart showing the number of coding and noncoding RNA targets of Slf1p, Sro9p, and Lhp1p (Inada and Guthrie 2004) among the top 100 targets (according to log₂ enrichment). (D) Pearson correlation coefficients between RIP-enrichments and selected mRNA characteristics. Abbreviations are as follows: Len., the length of the 5′ UTR, 3′ UTR, open reading frame (ORF) and the poly(A) tail (polyA); ribo., ribosome occupancy; half-life, mRNA half-life; and abund., mRNA abundance.

FIGURE 4.

Association of mRNAs with Slf1p depends on a functional LAM. (A) Immunoblot analysis following RIPs with TAP-tagged Slf1 (left) and the Slf1 LAM-mutant (right) expressed in slf1Δ cells using the peroxidase-anti-peroxidase soluble complex (PAP; see Materials and Methods). Lanes are as follows: IP, input yeast extract; SN, supernatant after incubation of extracts with IgG beads; B, captured beads; E, eluate from the beads; and pB, beads after elution of proteins with SDS-EDTA. (B) Four independent RIPs were performed with WT (pSLF1) or LAM-mutant (pLAM-m) SLF1, and the indicated mRNAs were quantified in RIP eluates by real-time qRT-PCR with cycle threshold (Ct) values normalized to mitochondrial 21S rRNA. Depicted is the fold difference in the abundance between WT and LAM-mutant RIP eluates. P-values were calculated based on ΔCt-values (t-test). **P < 0.001. (C) Comparative analysis of mRNA levels in the extracts (=input) used for RIP affinity purifications by qRT-PCR (two biological replicates). Mitochondrial 21S rRNA was used for normalization and averaged data for LAM-m was set to 1 (y-axis). P-values were calculated based on ΔCt-values (t-test). *P < 0.01, **P < 0.001.

FIGURE 5.

Overexpression of SLF1 positively affects Slf1p mRNA target levels. (A) Histogram depicting the fraction (y-axis) of average Cy5/Cy3 fluorescence ratios from duplicate microarray experiments comparing RNA levels in SLF1 overexpressing cells with control cells (empty vector) after 6 h (upper scheme) and 24 h (lower scheme). The bin size for fractions (y-axis) is 0.1 log₂ ratios (x-axis). The black line depicts the distribution of Slf1p RNA targets defined from RIP-Chip experiments. The gray line represents nontargets. **P-value < 0.001, Mann-Whitney test. (B) Immunoblot analysis with peroxidase-anti-peroxidase soluble complex (PAP) to detect tagged Slf1p and monitor the expression after 6 and 24 h of induction. Two biological replicates are shown with R1 and R2. Zwf1p is a loading control. (C) Box plots depicting relative differences in mRNA levels of Slf1p nontargets (white) and targets (gray) among copper and oxidative stress–related genes after 24 h of SLF1 overexpression. Averaged relative changes of mRNA expression levels are indicated as log₂ ratios. **P-value = 0.0013 (Mann-Whitney test).

FIGURE 6.

Slf1p is localized to the cytoplasm. Immunostaining of Slf1-TAP and Sro9-TAP in WT (MEX67) and Mex67 mutant cells (mex67-5) at the permissive (30°C) and nonpermissive (37°C) temperatures. White arrows point to accumulated nuclear Sro9p. DNA was visualized by DAPI staining.

FIGURE 7.

Slf1p stabilizes specific mRNA targets. (A) Scheme of RNA decay measurements. Transcription was halted by the addition of thiolutin to slf1Δ cells expressing either WT (pSLF1) or mutant (pLAM-m) Slf1 as a control. (B) Difference of remaining mRNA levels (pSLF1/pLAM-m expressing cells) of Slf1p targets (black) and nontargets (white) at various time points in minutes after thiolutin addition. Representative experiments are shown with standard deviations from three replicate measurements.