Sla1, a Schizosaccharomyces pombe homolog of the human La protein, induces ectopic meiosis when its C terminus is truncated

Abstract

Sla1 is a Schizosaccharomyces pombe homolog of the human La protein. La proteins are known to be RNA-binding proteins that bear conserved RNA recognition motifs (La and RRMs), but their biological functions still have not been fully resolved. In this study, we show that the S. pombe La homolog (Sla1) is involved in regulating sexual development. Sla1 truncated in the C terminus (Sla1DeltaC) induced ectopic sporulation in the ras1Delta strain and several other sporulation-deficient mutants. The C terminus contains a nuclear localization signal. While full-length Sla1 localizes in the nucleus, Sla1DeltaC is found throughout the cell, suggesting the cytoplasmic localization of Sla1DeltaC is involved in its sporulation-inducing activity. Further deletion analysis of Sla1 indicated that a small region (35 amino acids) that includes a portion of RRM2 is sufficient to induce sporulation. The La motif (RRM1) is not involved in this activity. Strikingly, Sla1DeltaC induced haploid meiosis in a heterothallic strain, similar to the pat1-114 or mei2-SATA mutation. Sla1DeltaC induced sporulation in a mei3 disruptant but not in a mei2 disruptant, indicating that Sla1DeltaC requires Mei2 to induce haploid meiosis. Deletion of the chromosomal sla1 gene lowered the temperature sensitivity of the pat1-114 mutant. Two-hybrid analysis indicated that Pat1 interacts with Sla1DeltaC but not full-length Sla1. Thus, Sla1DeltaC may block Pat1 activity. This block would remove the inhibition on Mei2, which would then drive the cell into haploid meiosis. Finally, Sla1 was degraded prior to the start of meiosis when we monitored Sla1 in cells in which meiosis was synchronously induced. The ability of truncated Sla1 to induce ectopic meiosis represents a very novel function that has hitherto not been suspected for the La family of proteins.

FIG.1.

Structure of the sla1⁺ gene. (A) Restriction map, subcloning, and construction of null mutants. Restriction enzyme sites: S, SacI; E, Eco T221; H, HincII. Spo indicates the effect of transforming the ras1Δ diploid strain with the subclones. (B) Comparison of the amino acid sequences of Sla1, human La, and S. cerevisiae Lhp1. Identical amino acids are marked with asterisks. The La protein-specific motif (RRM1) is shaded. RRM2 is shown in white against black. The putative NLS of Sla1 is indicated in boldface. The presumable basic bipartite NLS of human La is underlined. S.p., S. pombe; S.c., S. cerevisiae; H.s., Homo sapiens.

FIG. 2.

Sla1 localizes in the nucleus. (A) Strategy used to construct the genomic sla1-gfp fusion gene. (B) Subcellular localization of Sla1 tagged with GFP. NOR 12 was grown in PM (+N) minimal medium and shifted to PM (−N) medium to induce meiosis. Cells right after the shift (mitotic cell, +N) and cells incubated on PM (meiotic cell, −N) were examined for GFP fluorescence. Cells were counterstained with the DNA-binding dye Hoechst 33342 or DAPI to observe their nuclei. (C) Western blot analysis of Sla1-GFP. The Sla1-GFP proteins from NOR 1 cells and the wild-type (W.T.) strain harboring the plasmid were detected without degradation.

FIG. 3.

C-terminally truncated Sla1 and RRM2 induce sporulation in S. pombe TW5. The effect on sporulation of transforming the TW5 strain with constructs expressing Sla1 or a truncated variant is shown. The TW5 (h⁹⁰/h⁹⁰ ras1Δ/ras1Δ sla1Δ /sla1Δ) strain was transformed with the vector pSLF173L, with pSLF173L-Sla1FL (which overexpress sla1⁺), or with various pSLF173L constructs encoding truncated Sla1. The transformants were streaked on PM (+N) medium and incubated for 72 h at 25°C, and the percentage of sporulated cells was determined by counting at least 1,000 cells.

FIG. 4.

Subcellular localization of full-length or truncated Sla1-GFP proteins by live observation of cells. GFP fusion proteins were expressed under control of the nmt1 promoter in the vector pSLF172L-GFP. h⁹⁰ wild-type cells (SP870) carrying each plasmid were grown in PM (+N) medium and incubated at 25°C. Cells were examined for GFP fluorescence, and the same cells were stained with the DNA-binding dye Hoechst 33342. Images of the cells are depicted on the right.

FIG. 5.

Sla1ΔC induces haploid meiosis in the h⁻ wild-type strain. (A) h⁻ wild-type (W.T.) strain HM123 was transformed with vector pREP1, pREP1-Sla1ΔC, and pREP1-Sla1RRM. The transformants were streaked on PM (+N) medium and incubated for 72 h at 25°C, and the percentage of sporulated cells was determined by counting at least 1,000 cells. (B) Cell morphology of the haploid meiosis induced in HM123 due to Sla1ΔC expression. The plasmid pREP1-Sla1ΔC or pREP1-Sla1RRM caused the haploid cells to undergo a meiotic catastrophe. The resulting zygotes, including the spore-like bodies, are shown.

FIG. 6.

Yeast two-hybrid assay. S. cerevisiae strain L40 was transformed with plasmids harboring Sla1FL, Sla1ΔC, or Pat1 fused with the DNA binding domain or the DNA activating domain. The β-galactosidase activity (in nanomoles per minute per milligram of protein) of the resulting transformants was assayed by using o-nitrophenyl-β-d-galactopyranoside.

FIG. 7.

Lowering of the temperature sensitivity of the pat1-114 mutant by sla1 deletion. (Upper panel) Fivefold serial dilutions of pat1-114, pat1-114 sla1Δ, and sla1Δ cultures were spotted on YES medium and incubated at 25, 28, 30, 32.5, and 34°C. Two independently isolated strains with different mating type were tested. (Lower panel) The pat1-114 sla1Δ strain was transformed with pWH5 or pWH5-sla1⁺. Fivefold serial dilution of transformants were spotted on PMA medium and incubated at 25, 28, 30, and 32.5°C.

FIG.8.

Degradation of the Sla1 protein during meiosis. (A) Synchronous meiosis in the S964 strain. Left panel, DNA content measured by FACS analysis. Right panel, meiotic progression was monitored by DAPI staining of nuclei. The peaks of meiosis I (M I), meiosis II (M II), and spore formation (sporulation) are indicated. (B) Northern blot (N.B.) analysis of sla1⁺ expression. The leu1 mRNA level is shown as an internal control. veg, vegetative growing cell; Δ, sla1 disruptant. (C) Western blot analysis of Sla1 production. Cdc2 is present as an internal control. Arrowheads indicate truncated versions of Sla1p.