Ribosomal protein and biogenesis factors affect multiple steps during movement of the Saccharomyces cerevisiae Ty1 retrotransposon

Abstract

Background: A large number of Saccharomyces cerevisiae cellular factors modulate the movement of the retrovirus-like transposon Ty1. Surprisingly, a significant number of chromosomal genes required for Ty1 transposition encode components of the translational machinery, including ribosomal proteins, ribosomal biogenesis factors, protein trafficking proteins and protein or RNA modification enzymes.

Results: To assess the mechanistic connection between Ty1 mobility and the translation machinery, we have determined the effect of these mutations on ribosome biogenesis and Ty1 transcriptional and post-transcriptional regulation. Lack of genes encoding ribosomal proteins or ribosome assembly factors causes reduced accumulation of the ribosomal subunit with which they are associated. In addition, these mutations cause decreased Ty1 + 1 programmed translational frameshifting, and reduced Gag protein accumulation despite at least normal levels of Ty1 mRNA. Several ribosome subunit mutations increase the level of both an internally initiated Ty1 transcript and its encoded truncated Gag-p22 protein, which inhibits transposition.

Conclusions: Together, our results suggest that this large class of cellular genes modulate Ty1 transposition through multiple pathways. The effects are largely post-transcriptional acting at a variety of levels that may include translation initiation, protein stability and subcellular protein localization.

Keywords: Host factors; Programmed frameshifting; Retrotransposition; Ribosomal protein insufficiency; Ribosome biogenesis.

Fig. 1

Most translation-associated cofactor mutants show reductions in the relevant ribosomal subunit. Sucrose gradient analyses for wild type (WT) and mutant strains. The Y-axis represents the absorbance at 260 nm (A₂₆₀), proportional to RNA concentration, and the X-axis denotes increasing sucrose concentration with the lightest species eluting first (7–50 % sucrose). The 40S, 60S subunits, 80S monosome and polysomes in each of the profile are labeled and the presence of halfmers is denoted (black arrowhead)

Fig. 2

Analysis of translation-associated cofactor mutants for Ty1 + 1 programmed frameshift efficiency. The frameshift activity was measured using a β-galactosidase construct. The percent frameshift efficiency is reported as the activity of the frameshift construct relative to the frame fusion control construct. The unshaded column represents the frameshift activity for the wild type (WT) strain BY4741. The assays were repeated at least three times, each assay performed in triplicate. Asterisks indicate the frameshift activities of deletion strains that were significantly different from wild type activity as measured using ANOVA followed by the Tukey’s test (one asterisk, P ≤ 0.05; two asterisks P ≤ 0.005). Error bars represent the SEM. The ratio of the frameshift efficiency to the wild type appears above each column

Fig. 3

Ty1 Gag protein expression in various mutants defective in ribosomal biogenesis. Total cell protein was extracted from wild type (WT) and mutant strains, and subjected to Western blot analysis (50 μg/lane) using anti-VLP (upper panel) and control Hts1p (histidyl-tRNA synthetase; lower panel) antibodies. The Gag precursor (p49) and altered forms of Gag (Gag^†), which co-migrate under these electrophoretic conditions, and mature (p45) protein are indicated. Hts1p was used as a loading control. The extent of antibody reaction to both p49/Gag^† and p45 species of Gag, quantified using ImageJ, is shown beneath each cofactor mutant strain expressed as a ratio to that of the wild type strain. LSU = large (60S) ribosomal subunit; SSU = small (40S) ribosomal subunit

Fig. 4

Subunit-specific changes in the steady-state level of Ty1 mRNA by cofactor mutants. Total cellular RNA was hybridized with an [³²P]-labeled DNA probe specific to the mRNA encoding the RT domain of the Ty1 element (upper panel) or PYK1 control transcript (lower panel), which serves as a lane to lane control for the amount of RNA in the samples. The intensity of hybridization by the Ty1 probe relative to the PYK1 probe was quantified using the ImageJ software. The asterick (*) shows the position of the internally initiated Ty1i RNA detected in some of the mutants. LSU = large (60S) ribosomal subunit; SSU = small (40S) ribosomal subunit

Fig. 5

Ty1i RNA and Gag-p22/p18 expressed in ribosomal protein deletions. a Northern blot of total RNA from wild type (WT) and four mutant strains (spt3∆, rpl27A∆, rpl21A∆, rps0B∆ and rpl39). A ³²P-labeled riboprobe of Ty1 GAG (nt 1266-1601) hybridized to with full-length Ty1 mRNA (“Ty1”) and, especially in the spt3∆ mutant strain, to the subgenomic inhibitory Ty1i mRNA (“Ty1i”). b A Northern blot of poly(A)⁺ mRNA from the same strains. The same probe hybridized to Ty1 and Ty1i mRNAs. c Total protein extracts from the same strains was immunoblotted with a p18-specific antiserum to detect p49/p45/Gag^† and p22/p18. The histidyl-tRNA synthetase (Hts1) served as a loading control

Fig. 6

Distribution of Ty1 cofactor ribosomal proteins on the structure of the S. cerevisiae ribosome. The structure of the yeast ribosome [69] is derived from structure files 3J78 deposited in the Protein Data Bank (PDB; http://www.rcsb.org/pdb/) [70]. The structure was modeled using the VMD Molecular Graphics Viewer (http://www.ks.uiuc.edu/Research/vmd/) [71]. The structure of the rRNAs are shown as a surface with the large subunit rRNAs colored blue and the small subunit rRNA colored cyan. The ribosomal proteins are shown in cartoon mode with those encoded by Ty1 cofactor genes in red (large subunit) or pink (small subunit) and all others in cyan (large subunit) or blue (small subunit). a The 80S subunit seen from A site side. b The 80S subunit rotated 180° to view from the E site side. c The 60S subunit showing the surface that is in contact with the 40S in the 80S complex. d The 40S subunit showing the 60S interface surface. e The end of the nascent peptide channel from the peptide exits showing Rps39 (in white) located immediately inside the end on the right side of the exit channel; beyond Rps39, deep within the exit channel, the tip of a loop on Rps4 can be seen (in red)