Zebrafish RNase T2 genes and the evolution of secretory ribonucleases in animals

Abstract

Background: Members of the Ribonuclease (RNase) T2 family are common models for enzymological studies, and their evolution has been well characterized in plants. This family of acidic RNases is widespread, with members in almost all organisms including plants, animals, fungi, bacteria and even some viruses. While several biological functions have been proposed for these enzymes in plants, their role in animals is unknown. Interestingly, in vertebrates most of the biological roles of plant RNase T2 proteins are carried out by members of a different family, RNase A. Still, RNase T2 proteins are conserved in these animals

Results: As a first step to shed light on the role of animal RNase T2 enzymes, and to understand the evolution of these proteins while co-existing with the RNase A family, we characterized RNase Dre1 and RNase Dre2, the two RNase T2 genes present in the zebrafish (Danio rerio) genome. These genes are expressed in most tissues examined, including high expression in all stages of embryonic development, and their expression corresponds well with the presence of acidic RNase activities in every tissue analyzed. Embryo expression seems to be a conserved characteristic of members of this family, as other plant and animal RNase T2 genes show similar high expression during embryo development. While plant RNase T2 proteins and the vertebrate RNase A family show evidences of radiation and gene sorting, vertebrate RNase T2 proteins form a monophyletic group, but there is also another monophyletic group defining a fish-specific RNase T2 clade.

Conclusion: Based on gene expression and phylogenetic analyses we propose that RNase T2 enzymes carry out a housekeeping function. This conserved biological role probably kept RNase T2 enzymes in animal genomes in spite of the presence of RNases A. A hypothetical role during embryo development is also discussed.

Figure 1

Characterization of zebrafish RNases. A) Ribonuclease activities present in zebrafish extracts. Adult zebrafish extracts were analyzed in an in gel RNase assay at two different pHs. Adult zebrafish of mixed sexes were separated into "body" (B, mostly muscle, skin and skeleton), "head" (H, which included skull, muscle, skin, brain, eyes among other tissues) and "gut" (G, which included most internal organs such as gut, liver, sexual organs, heart). The size range for RNase T2 and RNase A proteins is indicated. B) Same samples as in A, analyzed by SDS-PAGE and stained with Coomassie Blue. One hundred μg of protein per lane were analyzed in both types of gels.

Figure 2

The zebrafish genome contains two RNase T2 genes. A) Alignment of the two predicted RNase T2 proteins (RNase Dre1 and RNase Dre2) present in the zebrafish genome with RNase T2 proteins from salmon (RNase Ok2), human (RNASET2), Arabidopsis thaliana (RNS1), tomato (RNase LE) and Aspergillus oryzae (RNase T2). Residues conserved in all RNase T2 enzymes are highlighted. CAS I and CAS II, conserved active-site segments that contain the two Histidines (*) involved in catalysis. The predicted signal peptides for RNase Dre1 and RNase DRe2 are underlined, and the alternative starting Methionine in RNase Dre1 is double-underlined. B) Structure of the two RNase T2 genes identified in the zebrafish genome. The intron-exon structure was obtained by comparison of the sequences obtained from direct cloning and sequencing of cDNA with the publish sequence of genomic DNA. Boxes indicate exons, lines indicate introns. Gray shading indicates untranslated regions, white indicates coding region, and black marks the region that undergoes alternative splicing in RNase Dre1. Gene accession numbers for the zebrafish proteins are FJ460212 for RNase Dre2 and FJ460210 and FJ460211 for the two different splicing variants of RNase Dre1.

Figure 3

Expression of zebrafish RNase Dre1 and RNase Dre2. A) RT-PCR analysis of expression of RNase Dre2 and RNase Dre1 in adult tissues: B, brain; E, eye; H, heart; L, liver; G, gut; M, muscle; O, ovary; T, testis; S, skin. p70 was used as control for loading. B) RT-PCR analysis of expression of RNase Dre2 and RNase Dre1 in embryos at different times (in days) after fertilization. C) Ribonuclease activities present in zebrafish embryos (E) and adults (A) analyzed by in gel activity assay as in Figure 1.

Figure 4

Localization of RNase Dre1 and RNase Dre2 expression in zebrafish embryos. Whole-mount in situ hybridization analysis was performed in embryos at the 1-cell stage (A, B), 16-cell stage (C, D) and prim 6 stage (E, F). Left panels, RNase Dre2 probe; right panels, RNase Dre1 probe.

Figure 5

RNase T2 genes are expressed in embryos in other organisms. Expression of RNase T2 genes during embryo development in the nematode Caenorhabditis elegans (A) and the plant Arabidopsis thaliana (B). Expression data were obtained from public microarray databases. Values indicate arbitrary fluorescence intensity units after normalization. A) Stages of nematode embryo development indicated as minutes after fertilization. B) Arabidopsis embryo stages: 1, globular; 2, heart; 3, triangle; 4, torpedo; 5, curly cotyledon; 6, curly cotyledon 2; 7, mature cotyledon; 8, green cotyledon.

Figure 6

Phylogenetic relationships of fish RNase T2 proteins, other animal RNase T2 proteins and bacterial, fungal and plant RNase T2s. Unrooted tree was obtained by the Neighbor-Joining method using only conserved regions. Bootstrap percentages (for 1,000 replications) greater than 50 are shown on interior branches. Color boxes highlight the clades that include fish RNases. Green indicates canonical CAS II, while yellow and red indicate mutations that putatively attenuate RNase activity (see figure 7). RNase Dre1 and RNase Dre2 are indicated with arrows.

Figure 7

Mutations in CAS II of fish RNase T2 proteins. The alignment shows the conserved CAS II region characteristic of RNase T2 enzymes. The absolutely conserved His (black box, white font) is part of the catalytic site of the enzyme. A second His residue (green), possibly involved in substrate binding or stabilization of an intermediate in the catalytic reaction, is mutated in most fish RNases. In the RNase Dre2 clade this His is mutated to Tyr (yellow). In the RNase Dre1 clade the His is mutated to a series of polar amino acids (red). Similar mutations are found in some plant S-RNases (S2 RNase and S3 RNase).

Figure 8

Hypothetical model of RNase T2 evolution in animals. An ancestral RNase T2 gene present in the last common ancestor of lancelet and higher chordate was duplicated after the separation of these two groups, but before the separation of Chondrichthyes and Teleostomi (black circle). Sometime after this duplication event RNase A genes emerged, most likely after the separation of Chondrichthyes and Teleostomi. The presence of RNase A could have released some selective pressure on RNase T2 genes, allowing the fixation of mutations in the active site conserved region (squares, H/Y/E-D position in CAS II), and the disappearance of one of the genes in tetrapods (black circle with white X).