Paralogous ribosomal protein l32-1 and l32-2 in fission yeast may function distinctively in cellular proliferation and quiescence by changing the ratio of rpl32 paralogs

Abstract

Fission yeast cells express Rpl32-2 highly while Rpl32-1 lowly in log phase; in contrast, expression of Rpl32-1 raises and reaches a peak level while Rpl32-2 is downregulated to a low basic level when cells enter into stationary phase. Overexpression of Rpl32-1 inhibits cell growth while overexpression of Rpl32-2 does not. Deleting rpl32-2 impairs cell growth more severely than deleting rpl32-1 does. Cell growth impaired by deleting either paralog can be rescued completely by reintroducing rpl32-2, but only partly by rpl32-1. Overexpression of Rpl32-1 inhibits cell division, yielding 4c DNA and multiple septa, while overexpressed Rpl32-2 promotes it. Transcriptomics analysis proved that Rpl32 paralogs regulate expression of a subset of genes related with cell division and stress response in a distinctive way. This functional difference of the two paralogs is due to their difference of 95(th) amino acid residue. The significance of a competitive inhibition between Rpl32 paralogs on their expression is discussed.

Figure 1. Expression of Rpl32 paralogs varied with different nutrient conditions during cultivation.

(A) The growth curve of WT cells (upper panel), and changes of mRNA level (upper panel) and protein level (lower panel) of Rpl32 paralogs in WT cells during cultivation. (B) mRNA level (upper panel) and protein level (lower panel) of Rpl32 paralogs in WT cells cultured in fresh EMM2, cell-free SP EMM2, cell-free LP EMM2, EMM2-N or EMM2-C medium respectively. QPCR was used for analysis of transcription level standardized with ACT1. Western blot was used for analysis of protein level and β-actin was an internal control.

Figure 2. Clustal alignment and nuclear localization signal sequence analysis of Rpl32 paralogs.

(A) Clustal alignment between Rrpl32-1 and Rpl32-2. Identical residues shared by these two paralogs are shaded black. (B) Localization of DAPI fluoresence (left), EGFP fluoresence (middled) and merged fluoresece (right) in rpl32-1-egfp,rpl32-2-egfp, rpl32-1-23-egfp or rpl32-2-23-egfp cells in log phase. Scale bar: 10 µm.

Figure 3. Overexpression or deletion of Rpl32 paralogs affected cell growth and their counterpart expression.

(A) Growth curve of WT, rpl32-1 or rpl32-2 cells. (B) Growth curve of WT, rpl32-1M or rpl32-2M cells. (C) mRNA level of Rpl32 paralogous genes in WT, rpl32-1, rpl32-2, rpl32-1M or rpl32-2M cells in log or early stationary phase, tested by QPCR standardized with ACT1. (D) Protein level of Rpl32-1 in rpl32-1-6his, rpl32-1-6his/rpl32-2 or rpl32-1-6his/rpl32-1M cells, Rpl32-2 in rpl32-2-HA, rpl32-2-HA/rpl32-1 or rpl32-1-6his/rpl32-2M cells, and total Rpl32 in WT, rpl32-1, rpl32-2, rpl32-1M or rpl32-2M cells in log or stationary phase, tested by Western blot. β-actin was used as internal control. (E) Protein level of total Rpl32 in rpl32-1△, rpl32-2△, rpl32-1△/rpl32-1, rpl32-1△/rpl32-2, rpl32-2△/rpl32-1, rpl32-2△/rpl32-2, or WT cells in log phase, tested by Western blot. β-actin was used as internal control. (F) Growth curve of rpl32-1△, rpl32-2△, rpl32-1△/rpl32-1, rpl32-1△/rpl32-2, rpl32-2△/rpl32-1, rpl32-2△/rpl32-2, or WT cells.

Figure 4. Cellular analysis of cells overexpressing Rpl32 paralogs.

(A) DNA component in rpl32-1, rpl32-2, rpl32-1M, rpl32-2M, or WT cells in log or stationary phase, tested by FACS. 2C indicates the DNA content in a cell containing one G2 nucleus or a mitotic cell containing two G1 nuclei. 4C indicates the DNA content in a cell containing two G2 nuclei. (B) DIC and DAPI images of cells in (A) in log or stationary phase. Arrow indicates defect in nuclei division. scale bar: 10 µm. (C) Nuclei counting of cells from (B) in stationary phase. 300 cells per sample were counted. (D) CW images of septa in cells from (A) in log phase and stationary phase. Arrow indicates multiple septa or septa-like stucture. Scale bar: 10 µm. (E) Quantification of the percentage of septa in cells in (D) in log phase. 300 cells per sample were counted. (F) DNA component in cells synchronized with HU in (A) duiring elutriation, analysed with FACS.

Figure 5. Transcriptomic patterns of WT, rpl32-1 or rpl32-2 cells respectively in log or stationary phase.

Color panel indicates relative increase (red), decrease (blue) and median (white) of transcription level for 64 genes regulated by Rpl32-1 and Rpl32-2 in a distinctive way (at least 2-fold changes). Bold type means genes are regulated oppositely by both Rpl32 paralogs (Fold change >2) and nonbold type means genes are regulated by either of Rpl32 paralogs (Fold change >2). * Changes in transcripts level were confirmed by QPCR.

Figure 6. Schematic representation of a possible regulation of cell growth states by changing the ratio of Rpl32 paralogs.

In rich nutrient environment, cells highly express Rpl32-2 while lowly express Rpl32-1 to promote cell division for proliferation. Nutrition deficiency induces cells to upregulate Rpl32-1 expression and downregulate Rpl32-2 expression to inhibit cell division for quiescence.