Elsevier

Gene

Volume 373, 24 May 2006, Pages 116-125
Gene

Ancient expansion of the ribonuclease A superfamily revealed by genomic analysis of placental and marsupial mammals

https://doi.org/10.1016/j.gene.2006.01.018Get rights and content

Abstract

Members of the ribonuclease (RNase) A superfamily participate in a diverse array of biological processes, including digestion, angiogenesis, innate immunity, and possibly male reproduction. The superfamily is vertebrate-specific, with 13–20 highly divergent members in primates and rodents, but only a few members in chicken and fish. This has led to the proposal that the superfamily started off from a progenitor with structural similarities to angiogenin and that the suerfamily underwent a dramatic expansion during mammalian evolution. To date this evolutionary expansion and understand the functional diversification of the superfamily, we here determine its entire repertoire in the sequenced genomes of dog, cow, and opossum. We identified 7, 20, and 21 putatively functional RNase genes from these three species, respectively. Many of the identified genes are highly divergent from all previously known RNase genes, thus representing new lineages within the superfamily. Phylogenetic analysis indicates that the superfamily expansion predated the separation of placental and marsupial mammals and that differential gene loss and duplication occurred in different species, generating a great variation in gene number and content among extant mammals.

Introduction

Represented by the famous prototype, bovine pancreatic ribonuclease (RNase A), the RNase A superfamily is one of the best studied protein families. Numerous fundamental discoveries have been made in biochemistry, molecular biology, structural biology, enzymology, and molecular evolution by studying various members of the superfamily (Anfinsen, 1973, Blackburn and Moore, 1982, Jermann et al., 1995, Rosenberg et al., 1995, D'Alessio and Riordan, 1997, Beintema and Kleineidam, 1998, Zhang et al., 1998, Zhang et al., 2002b, Cho et al., 2005). Note that some unrelated proteins, such as RNases H, L, P, and T, and also ribozymes, have the ribonucleolytic activity, because this activity originated multiple times in evolution. However, for simplicity, we use “RNase” to refer to the RNase A superfamily or its members in this article. RNases are known to have a diverse array of physiological functions. For example, pancreatic ribonuclease (RNase 1) degrades dietary RNA molecules in the digestive gut (Barnard, 1969), angiogenin (RNase 5) stimulates blood vessel formation (Fett et al., 1985, Strydom et al., 1985), and RNases 2, 3, and 7 have antibacterial or antiviral activities that are believed to be used in innate immunity (Young et al., 1986, Domachowske et al., 1998a, Domachowske et al., 1998b, Harder and Schroder, 2002, Zhang and Rosenberg, 2002a, Zhang et al., 2003). RNases share a number of common sequence features. The entire open reading frame (ORF) of ∼ 130 codons is encoded in a single exon. RNases have a signal peptide at the N-terminus and six to eight conserved cysteines that form disulfide bridges. They have three distinct catalytic residues, called the catalytic triad, and several other conserved motifs, although the level of conservation varies among proteins (Beintema and Kleineidam, 1998, Cho et al., 2005). To date, 13 human RNase genes have been reported (Cho et al., 2005). Eight of them have been subject to functional characterizations and are known to have the ribonucleolytic activity. These RNases are the pancreatic ribonuclease (RNase 1), eosinophil-derived neutotoxin (EDN or RNase 2), eosinophil cationic protein (ECP or RNase 3), RNase 4, angiogenin (RNase 5), RNase 6 (or k6), RNase 7, and RNase 8. RNases 9–13 were identified only recently and RNases 9 and 10 have no detectable RNase activities, while RNase activities have not been examined for RNases 11–13 (Penttinen et al., 2003, Castella et al., 2004a, Castella et al., 2004b, Devor et al., 2004, Cho et al., 2005). Except for RNases 2 and 3 and RNases 7 and 8, which emerged by gene duplication within primates (Rosenberg et al., 1995, Zhang et al., 2002a, Zhang et al., 2003), all human RNase genes are represented (in one or multiple copies) in the mouse and rat (Cho et al., 2005). In the chicken and zebrafish genomes, however, only a limited number of closely related RNase genes are found (Cho et al., 2005). In the bullfrogs, although several divergent RNases have been reported, they are more closely related to each other than to any mammalian RNases, thus representing an amphibian-specific cluster (Rosenberg et al., 2001). RNases have never been found in any invertebrates, including the genomes of two completely sequenced urochordates, Ciona intestinalis and Ciona savignyi (Cho et al., 2005). Evolutionary analysis and structural features indicate that angiogenins are the first diverging group in mammalian RNases (Cho et al., 2005) with all non-mammalian vertebrate RNases having angionenin-like structures, although the angiogenic activity has yet to be examined or detected in these RNases. The phylogenetic patterns suggest that the superfamily arose in early vertebrate evolution as an angiogenin-like molecule and it underwent independent expansions during amphibian evolution and mammalian evolution. There are several interesting questions with regard to the mammalian expansion of the RNase superfamily. First, a previous study (Cho et al., 2005) showed that the expansion occurred after the bird–mammal divergence [∼ 310 My ago; (Hedges et al., 1996)], but before the primate–rodent separation [∼ 85 My ago; (Murphy et al., 2004)], leaving the precise date of the expansion uncertain. Second, previous studies were limited to primates and rodents, with the size and diversity of the RNase superfamily unknown in any other orders of placental mammals or any non-placental mammals. The draft genome sequences of the dog (Canis familiaris), cow (Bos taurus), and opossum (Monodelphis domestica) became available recently, providing an opportunity to address the above questions. As a marsupial, the opossum is particularly valuable for unraveling the timing of the mammalian RNases superfamily expansion. In this work, we identify all RNase genes from the above three genome sequences and report that the mammalian RNase superfamily expanded before the split between placental and marsupial mammals [∼ 180 My ago; (Murphy et al., 2004)]. We further show that this expansion was followed by differential gene retention and duplication among different orders of placental mammals, generating a great variation in the RNase gene repertoire among species.

Section snippets

Materials and methods

In this paper, we use “dog” for C. familiaris (Cf for short), “cow” for B. taurus (Bt), and “opossum” for the gray short-tailed opossum M. domestica (Md). A “functional gene” refers to an RNase gene that contains an uninterrupted open reading frame (ORF), whereas a “pseudogene” refers to a gene whose ORF is interrupted by a premature stop codon anywhere in the ORF or by frame-shifting insertions/deletions. Pseudogenes are distinguished from functional genes by having “ps” after the gene name

Identification of RNase A genes in dog, cow, and opossum

From whole-genome searches based on TBLASTN, we identified RNase genes and pseudogenes from the genome sequences of the dog, cow, and opossum. We also conducted BLASTN-based nucleotide sequence searches to identify pseudogenes that had not been found by TBLASTN. The entire catalogs of the RNase genes identified in this study are listed in Table 1 and Supplementary Tables S1–S3, and the DNA sequences of the RNase genes are provided in Supplementary Data Sets 1–3. The chromosomal locations of the

Acknowledgments

We thank Jaap Beintema for valuable comments and Eric Devor for sharing unpublished data. This work was supported by National Institutes of Health grant GM67030 to J.Z.

References (45)

  • L.F. Cadavid et al.

    Evolutionary instability of the major histocompatibility complex class I loci in New World primates

    Proc. Natl. Acad. Sci. U. S. A.

    (1997)
  • S. Castella et al.

    Train A, an RNase a-like protein without RNase activity, is secreted and reabsorbed by the same epididymal cells under testicular control

    Biol. Reprod.

    (2004)
  • S. Castella

    Identification of a member of a new RNase a family specifically secreted by epididymal caput epithelium

    Biol. Reprod.

    (2004)
  • E. Confalone

    Molecular evolution of genes encoding ribonucleases in ruminant species

    J. Mol. Evol.

    (1995)
  • J. D'Alessio et al.

    Ribonucleases: Structures and Functions

    (1997)
  • E.J. Devor et al.

    LOC 390443 (RNase 9) on chromosome 14q11.2 is related to the RNase a superfamily and contains a unique amino-terminal preproteinlike sequence

    Hum. Biol.

    (2004)
  • J.B. Domachowske et al.

    Eosinophil cationic protein RNase 3 is another RNaseA-family ribonuclease with direct antiviral activity

    Nucleic Acids Res.

    (1998)
  • J.B. Domachowske et al.

    Recombinant human eosinophil-derived neurotoxin/RNase 2 functions as an effective antiviral agent against respiratory syncytial virus

    J. Infect. Dis.

    (1998)
  • K.D. Dyer et al.

    The mouse RNase 4 and RNase 5/ang 1 locus utilizes dual promoters for tissue-specific expression

    Nucleic Acids Res.

    (2005)
  • J. Felsenstein

    Confidence-limits on phylogenies—an approach using the bootstrap

    Evolution

    (1985)
  • J.W. Fett

    Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma-cells

    Biochemistry

    (1985)
  • S.B. Hedges et al.

    Continental breakup and the ordinal diversification of birds and mammals

    Nature

    (1996)
  • Cited by (0)

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