Ribosomal frameshifting in decoding antizyme mRNAs from yeast and protists to humans: close to 300 cases reveal remarkable diversity despite underlying conservation

Abstract

The protein antizyme is a negative regulator of intracellular polyamine levels. Ribosomes synthesizing antizyme start in one ORF and at the codon 5' adjacent to its stop codon, shift +1 to a second and partially overlapping ORF which encodes most of the protein. The ribosomal frameshifting is a sensor and effector of an autoregulatory circuit which is conserved in animals, fungi and protists. Stimulatory signals encoded 5' and 3' of the shift site act to program the frameshifting. Despite overall conservation, many individual branches have evolved specific features surrounding the frameshift site. Among these are RNA pseudoknots, RNA stem-loops, conserved primary RNA sequences, nascent peptide sequences and branch-specific 'shifty' codons.

Figure 1.

The phylogenetic relationship of a select number of fungal antizyme frameshift sites. The unrooted tree is based on the amino acid sequence (encoded by both ORF1 and ORF2) and was drawn using the ClustalX program neighbor-joining algorithm. Different colored lines indicate different frameshift sites. Bootstrap values are given for key nodes. Contaminant from plant library.

Figure 2.

2D representation of RNA pseudoknots—class I (A) of mouse antizyme 1 and (B) mouse antizyme 2 mRNAs. The frameshift site is indicated with orange letters. Black arrowheads represent substitutions deduced from phylogenetic comparison to orthologous genes. Non-compensatory changes in the stems are shown in black letters; compensatory changes are shown in blue letters.

Figure 3.

2D representation of RNA pseudoknots (A) class IIa—the actual sequence is of Crassostrea gigas (B) class IIb—the sequence is of an aphid. The frameshift site is indicated with orange letters. Black arrowheads represent substitutions deduced from phylogenetic comparison to orthologous genes. Non-compensatory changes in the stems are shown in black letters; compensatory changes are shown in blue letters.

Figure 4.

Newly identified antizyme mRNA potential 3′ stimulators. Three elements strongly supported by phylogenetic data are shown. The frameshift sites are indicated with orange letters. (A) 3′ conserved stem-loop element in a large subset of fungi. The actual sequence shown is of N. crassa. Base-paired nucleotides are in red. The loop is shown in green. Blue-colored nucleotides following the stem-loop are absolutely conserved. (B) Base-paired nucleotides are in red. Nucleotides potentially extending stem 2 by non-Watson–Crick base-pairs are shown in magenta. The loop is shown in green. Black arrowheads represent substitutions deduced from phylogenetic comparison to antizymes in other mushroom-related species. Non-compensatory changes in the stems are shown in black letters; compensatory changes are shown in blue letters; and a potential non-Watson–Crick G-A base-pairing nucleotides are shown in gray. The actual sequence shown is of Agaricus bisporus. (C) The conserved 3′ element in flies, mosquitoes and midges. The top line shows the nucleotide sequence and the bottom line the amino acid sequence. Only absolutely conserved positions are shown. Variant nucleotide positions are indicated by ‘-’. Alternating codons in the +1 frame (in-frame with ORF2) are shown in red and black.

Figure 5.

Conserved nucleotide sequences 5′ of the frameshift site. In each case only the consensus is shown and poorly conserved positions are indicated by ‘-‘. Gaps in alignments are shown as empty spaces. The frameshift site is highlighted in black. Module A is highlighted in red. Module B is highlighted in light blue. Module C is highlighted in gray. Module D is highlighted in yellow. Module E is highlighted in dark blue. Module F is highlighted in purple. ‘PK’ = RNA pseudoknot. *Flies, midges and mosquitoes. **C. elegans and related nematodes (see Figure S1C). ***Onchocerca volvulus and related nematodes (see Figure S1C).

Figure 6.

Conserved sequences 5′ of the frameshift sites of mushrooms and other Basidiomycota. The most highly conserved region is indicated by red letters. (A) Alignment of the 90 nt 5′ of the frameshift site of 10 mushroom-related species. Absolutely conserved positions are shown by ‘*’. Alternating codons in the 0 frame (in-frame with ORF1) are highlighted in light blue or not at all. (B) Alignment of the last 29 amino acids of ORF1 of 10 mushroom-related species. Absolutely conserved positions are shown by ‘*’. (C) Comparison of the last 15 amino acids of ORF1 of different Basidiomycota—mushrooms, Ustilago maydis and Cryptoccocus spp. The Cryptoccocus sequence is based on several C. neoformans strains and C. laurentii.

Figure 7.

Nucleotide alignment of conserved elements in the 3′ UTR of vertebrate antizyme genes. Gaps in alignment are shown by ‘-‘. The number of the top line in each case indicates the distance to the stop codon of ORF2 in human. (A) Alignment of the conserved region in orthologs of antizyme 1. Absolutely conserved nucleotides are in red and those conserved in at least 11 of the 14 species are in blue. Less well-conserved positions are in black. The two heptanucleotide sequences matching the consensus polyadenylation site are highlighted in green. (B) Absolutely conserved nucleotides are in red. Less well-conserved positions are in black. The upstream conserved region is highlighted in yellow. The downstream conserved region is highlighted in blue. The polyadenylation sites are highlighted in green. Species abbreviations are as follows: H.s. = human, M.m. = mouse, R.n. = rat, C.f. = dog, D.n. = nine-banded armadillo, E.c. = horse, B.t. = cow, O.a. = sheep, S.s. = pig, C.p. = guinea pig, E.t. = Madagascar hedgehog, M.d. = opossum, G.g. = chicken, T.g. = zebra finch.