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. 2011 Aug 28;43(10):948-55.
doi: 10.1038/ng.906.

Pol III binding in six mammals shows conservation among amino acid isotypes despite divergence among tRNA genes

Claudia Kutter  1 , Gordon D Brown, Angela Gonçalves, Michael D Wilson, Stephen Watt, Alvis Brazma, Robert J White, Duncan T Odom
Affiliations

Pol III binding in six mammals shows conservation among amino acid isotypes despite divergence among tRNA genes

Claudia Kutter et al. Nat Genet. .

Abstract

RNA polymerase III (Pol III) transcription of tRNA genes is essential for generating the tRNA adaptor molecules that link genetic sequence and protein translation. By mapping Pol III occupancy genome-wide in mouse, rat, human, macaque, dog and opossum livers, we found that Pol III binding to individual tRNA genes varies substantially in strength and location. However, when we took into account tRNA redundancies by grouping Pol III occupancy into 46 anticodon isoacceptor families or 21 amino acid-based isotype classes, we discovered strong conservation. Similarly, Pol III occupancy of amino acid isotypes is almost invariant among transcriptionally and evolutionarily diverse tissues in mouse. Thus, synthesis of functional tRNA isotypes has been highly constrained, although the usage of individual tRNA genes has evolved rapidly.

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Figures

Figure 1
Figure 1. Pol III occupies and transcribes tRNA genes in mouse liver
(A) tRNA genes are present in hundreds of copies in a mammalian genome, but collapse into 62 isoacceptor families (48 are present in mammals), and 21 amino acid isotypes. (B) Separate portions of each species’ tissues were flash frozen to permit direct RNA sequencing, and treated with formaldehyde to cross link protein-DNA contacts to allow chromatin immunoprecipitation reactions. (C) Primary tissues were isolated from six mammals varying in evolutionary distance to mouse, from 12 million (rat) to over 180 million years (opossum). (D) Typical pol III-bound tRNA loci on murine chromosome 11. Pol III binding shown as a beige enrichment track (top), the input DNA as a black track (middle), and total RNA as blue track (bottom), indicating the pol III bound regions are transcriptionally active. The y-axis of each track specifies read density. The mammalian conservation track, obtained from UCSC, displays the degree of placental mammal basewise conservation (20 species) and sequence constraint. Beneath is the genome annotation in this region obtained for repeats, non-coding RNAs, and tRNAs (UCSC genome browser).
Figure 2
Figure 2. tRNA isoacceptors and isotypes are differentially bound by pol III in mouse liver
(A) Pol III binding varies widely among isoacceptor families (red), which collapse in number as well as binding variability in the 21 amino acid isotypes (grey). Areas are shaded according to number of pol III read counts (dendrogram shows values white: low to red/black: high). Hatched boxes represent isoacceptors that are absent in mouse, or that do not encode for an amino acid. (B-D) Pol III binding between replicates of mouse liver compared at (B) tRNA gene loci (blue), (C) anticodon isoacceptors (red), and (D) amino acid isotypes (black). Spearman’s rank correlation coefficients (ρ) are shown.
Figure 3
Figure 3. Amino acid isotypes are bound by pol III in a tissue-independent manner
Pol III occupancy at tRNA genes was determined in mouse liver, muscle, and testes. The intersection of the row-column for each tissue combination in the upper-right triangle shows the correlation for all pol III bound tRNA gene loci (blue) and isoacceptor (red) and in the lower-left triangle of the binding of pol III to isotypes (black). The Spearman’s rank correlation coefficients (ρ) are reported correspondingly by colour in the lower right of each inter-tissue panel.
Figure 4
Figure 4. The tRNA genes bound by pol III diverge in genomic location and functional usage among mammals
(A) Two clusters of multiple tRNA genes flanking the Trim41 and Trim7 genes are bound by pol III in multiple species (shown as genome tracks). The numbers on the left of each track specify read density for pol III ChIP experiments. The syntenic position of each tRNA gene is traced between species with a dashed line. A blue dashed line represents conserved binding in all six mammals and a yellow line indicates species-specific innovation. The cluster upstream of the Trim41 gene shows four tRNA gene loci (two valines, a lysine, and an alanine) with conserved pol III binding. The lysine gene locus does not exist in the rodents, and two leucines are only present in opossum. An additional cluster 3′ downstream of Trim7 demonstrates two tRNA loci (threonine and proline) bound by pol III in all six species. The valine tRNA gene only evolved in the eutherians, and the leucine tRNA gene is not bound in dog. (B) A 5-way Venn diagram intersects the tRNA genes bound by pol III for each possible combination of eutherian mammals used in this study. The total number of species-specific tRNA genes (white) is subdivided into tRNA genes present (inner black numbers) and absent (outer black numbers in dashed subdivision) in Ensembl’s 16 amniote alignment. 24 tRNA genes are bound in all 6 species. Area colourings are shaded according to number of tRNA genes (white: low to blue: high). (C) tRNA loci bound by pol III from panel (B) were sorted by the number of species in which they were bound, and compared to the level of pol III occupancy in each of the study species; demonstrating that higher degree of tRNA gene conservation correlates with higher pol III occupancy (Methods).
Figure 5
Figure 5. Isotypes are highly conserved in pol III occupancy and codon usage across mammalian evolution, though the isoacceptors they consist of are only moderately well conserved
We experimentally determined the strength of pol III binding using ChIP-seq (A-C) and transcript abundance using mRNA-seq (D-F) in livers of six mammals. (A) Proportional frequency of pol III binding by isotype is similar among all six mammals. (B) Proportional frequency of pol III binding to all possible arginine tRNA isoacceptors demonstrate differences among mammals. (C) Pol III binding to tRNA anticodons diverges in correspondence to evolutionary distance (red circles), but pol III binding to isotypes remains largely conserved out to 180 MY of evolution (hollow circles). (D) The frequency of each amino acid weighted by transcript expression was highly conserved between species. (E) Proportional frequencies of codon usage for arginine indicate that mammalian species have little divergence in their codon biases. (F) Amino acids and to a large extent their encoding triplet codon are equally abundant in transcripts across 180 MY of mammalian evolution. Coloured lines within the radial plots (A, B, D, and E) present the data values of each species (purple: mouse, pink: rat, green: human, orange: macaque, black: dog, and yellow: opossum). Labels around the plot indicate amino acids (A and D), anticodons (B) and triplet codons (E). Labels within the grid of the radial plots (A, B, D, and E) describe percentages.
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