Human ribosomal protein S13 regulates expression of its own gene at the splicing step by a feedback mechanism

Abstract

The expression of ribosomal protein (rp) genes is regulated at multiple levels. In yeast, two genes are autoregulated by feedback effects of the protein on pre-mRNA splicing. Here, we have investigated whether similar mechanisms occur in eukaryotes with more complicated and highly regulated splicing patterns. Comparisons of the sequences of ribosomal protein S13 gene (RPS13) among mammals and birds revealed that intron 1 is more conserved than the other introns. Transfection of HEK 293 cells with a minigene-expressing ribosomal protein S13 showed that the presence of intron 1 reduced expression by a factor of four. Ribosomal protein S13 was found to inhibit excision of intron 1 from rpS13 pre-mRNA fragment in vitro. This protein was shown to be able to specifically bind the fragment and to confer protection against ribonuclease cleavage at sequences near the 5' and 3' splice sites. The results suggest that overproduction of rpS13 in mammalian cells interferes with splicing of its own pre-mRNA by a feedback mechanism.

Figure 1.

Intron 1 in RPS13 gene of mammals and birds is evolutionary conserved. Generated by the ECR browser (35) graphic plot of genomic alignment and comparative analysis with the genes coding for rpS13 in dog, mouse, rat and chicken. Smooth peaks represent levels of sequence identity. Exons coloured in blue, introns in pink. Intron 1 is marked by brace.

Figure 2.

Overexpression of rpS13 in mammalian cells suppresses the expression of mRNA from a minigene containing intron 1. (A) Diagrams of vectors pECFP-S13 and pECFP-S13-int1. (B) Relative amount of mRNA coding for rpS13 (both endogenous and plasmid-derived) in HEK 293 cells. Non-transfected cells (column 1), cells transfected with pECFP-N1 (column 2), cells transfected with pECFP-S13 (accepted as 100 arbitrary units, column 3) and cells transfected with pECFP-S13-int 1 (column 4). Experiments made in triplicate; error bars are shown. For standardization of the quantity of RNA in the samples primers specific to GAPDH cDNA were used. Dispersal of amount of GAPDH cDNA in the samples that reflects total amount of RNA was ±25% from average value.

Figure 3.

rpS13 preferentially binds to its RNA containing the first intron (S13INT). (A) Schematic diagram of human RPS13 gene showing the location of the exons (numbered shadowed boxes) and a scheme of S13INT synthesis. (B) Isotherms of binding of ³²P-labelled S13INT to rpS13 (filled circles), rpS16 (open circles) and rpS10 (filled triangles). (C) Displacement of ³²P-labelled S13INT from its complex with rpS13 by competition with unlabelled RNAs: S13INT (filled circles), AdML (open circles) and poly (AU) (filled triangles). (D) Isotherms of binding of various ³²P-labelled pre-mRNA fragments containing intron 1 to rpS13: S13INT (filled circles), S13INTm (open circles), rpS16 pre-mRNA fragment (filled triangles) and rpS26 pre-mRNA fragment (open triangles). (E) Immunoprecipitation of rpS13 associated with ³²P-labelled RNA in nuclear extract. The mixture of S13INT, rpS17 and rpS26 RNA was separated by denaturing PAGE after isolation on beads either charged with antibodies against rpS13 (lane 1), or uncharged (unspecific sorbtion, lane 2); lane 3, aliquot of the initial mixture; lane 4, aliquot of the sample containing RNAs unbound to the beads charged with antibodies. Columns illustrate the proportion of each RNA transcript in the initial mixture (‘before’) and in the mixture isolated from nuclear extract (‘after’).

Figure 4.

rpS13 specifically inhibits in vitro splicing of the S13INT transcript. (A) Effect of rpS13 on splicing of S13INT and rpS16 pre-mRNA fragment. Products of splicing were separated on 6% denaturing PAAG. Time course reactions of the transcripts alone (lanes 1–4 and 10–13) and 2 h incubation under splicing conditions of the transcripts taken together in the presence of rpS13 at concentrations of 0.3 μM (lane 6), 1 μM (lane 7), 3 μM (lane 8), 8 μM (lane 9) or without the protein (lane 5). Positions of RNAs on the gel and their lengths are denoted on left (for S13INT) and on right (for rpS16 pre-mRNA). Arrows on the gel (lane 9) indicate presence of the mRNA product for rpS16 pre-mRNA splicing and its absence for S13INT. (B) Effect of rpS13, rpS16 and rpS10 on the S13INT splicing. Products of S13INT splicing after 2 h incubation under splicing conditions in the presence of rpS13 (lanes 2–4), rpS10 (lanes 5–7) and rpS16 (lanes 8–10) at concentrations of 1 μM (lanes 2, 5, 8), 5 μM (lanes 3, 6, 9), 8 μM (lanes 4, 7, 10) or without protein (lane 1), separated on 6% denaturing gel. The positions of the RNAs are indicated on right. (C) Comparison of effects of rpS13 on the in vitro splicing of S13INT transcript and of the transcript mutated in the vicinity of both the 5′ and 3′ splice sites (S13INTm). Lanes 1–3 and 10–12—time-course reactions. Splicing products after 2 h incubation of S13INT (lanes 4–6) and S13INTm (lanes 7–9) under splicing conditions at the presence of rpS13 at the concentration 1 μM (lanes 4 and 7), 3 μM (lanes 5 and 8), 6 μM (lanes 6 and 9). Arrows indicate presence of the mRNA product with S13INTm (lane 9) and its absence with S13INT (lane 6). The diagram below represents quantification of the gel data. Each column represents the ratio of the intensities of bands for the mRNA product and the pre-mRNA on the respective lane on the gel in arbitrary units. The height of column is proportional to the splicing efficiency. White columns are for S13INT, grey ones are for S13INTm. Columns are grouped in pairs for splicing reactions carried out under the same conditions.

Figure 5.

rpS13 binds to S13INT in the vicinity of the splice sites. Footprinting of S13INT transcript in the complex with rpS13. (A) Ensemble centroid of S13INT calculated by Sfold 2.0 (38). Positions of rpS13 protection from RNase cleavage are shown by arrows marked with the respective nucleotide positions. Boxes with arrows indicate positions of the 5′ and 3′ splice sites. (B) Primer extension analysis of the S13INT transcript cleaved with RNAses T1, T2 and V1 free (−) or in the complex with rpS13 (+). Lanes A, C, G and T correspond to the sequencing reactions. Positions of the nucleotides protected by the protein from RNase cleavage (footprints) are marked by arrows. Primers complementary to positions 103–116 (panel 1) and 235–250 (panels 2 and 3) of S13INT transcript were used in the study.

Figure 6.

Schematic representation of possible pathways for autoregulation of the biosynthesis of rpS13 via splicing of intron 1.