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Evolution of a TRIM5-CypA splice isoform in old world monkeys.

Newman RM, Hall L, Kirmaier A, Pozzi LA, Pery E, Farzan M, O'Neil SP, Johnson W - PLoS Pathog. (2008)

Bottom Line: The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform.HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A.Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts, United States of America.

ABSTRACT
The TRIM family proteins share a conserved arrangement of three adjacent domains, an N-terminal RING domain, followed by one or two B-boxes and a coiled-coil, which constitutes the tripartite-motif for which the family is named. However, the C-termini of TRIM proteins vary, and include at least nine evolutionarily distinct, unrelated protein domains. Antiviral restriction factor TRIM5alpha has a C-terminal B30.2/SPRY domain, which is the major determinant of viral target specificity. Here, we describe the evolution of a cyclophilin-A encoding exon downstream of the TRIM5 locus of Asian macaques. Alternative splicing gives rise to chimeric transcripts encoding the TRIM motif fused to a C-terminal CypA domain (TRIM5-CypA). We detected TRIM5-CypA chimeric transcripts in primary lymphocytes from two macaque species. These were derived in part from a CypA pseudogene in the TRIM5 locus, which is distinct from the previously described CypA insertion in TRIM5 of owl monkeys. The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform. All pig-tailed macaques (M. nemestrina) screened were homozygous for the CypA insertion. In contrast, the CypA-containing allele was present in 17% (17/101) of rhesus macaques (M. mulatta). The block to HIV-1 infection in lymphocytes from animals bearing the TRIM5-CypA allele was weaker than that in cells from wild type animals. HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A. Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1. Despite its distinct evolutionary origin, Macaca TRIM5-CypA has a similar domain arrangement and shares approximately 80% amino-acid identity with the TRIMCyp protein of owl monkeys. The independent appearance of TRIM5-CypA chimeras in two primate lineages constitutes a remarkable example of convergent evolution. Based on the presence of the CypA insertion in separate macaque lineages, and its absence from sooty mangabeys, we estimate that the Macaca TRIM5-CypA variant appeared 5-10 million years ago in a common ancestor of the Asian macaques. Whether the formation of novel genes through alternative splicing has played a wider role in the evolution of the TRIM family remains to be investigated.

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A TRIM5-CypA transcript of macaques is generated by alternative splicing.A. Messenger RNA sequence spanning the junction between the TRIM5 exon-6 and CypA sequences. B. Predicted amino-acid sequence of rhesus TRIM5-CypA aligned with pig-tailed macaque TRIM5-CypA and owl monkey TRIMCyp. Residues that differ from rhesus TRIM5-CypA are highlighted in blue. The region encoded by exon-7 is boxed in red; as the result of alternative splicing, this sequence is present in owl monkey TRIMCyp, but is missing from old world monkey TRIM5-CypA. C. Cartoon depicting predicted protein sequences of the old world monkey TRIM5-CypA protein (top), the owl monkey TRIM-Cyp protein (middle) and wild type primate TRIM5α (bottom).
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ppat-1000003-g003: A TRIM5-CypA transcript of macaques is generated by alternative splicing.A. Messenger RNA sequence spanning the junction between the TRIM5 exon-6 and CypA sequences. B. Predicted amino-acid sequence of rhesus TRIM5-CypA aligned with pig-tailed macaque TRIM5-CypA and owl monkey TRIMCyp. Residues that differ from rhesus TRIM5-CypA are highlighted in blue. The region encoded by exon-7 is boxed in red; as the result of alternative splicing, this sequence is present in owl monkey TRIMCyp, but is missing from old world monkey TRIM5-CypA. C. Cartoon depicting predicted protein sequences of the old world monkey TRIM5-CypA protein (top), the owl monkey TRIM-Cyp protein (middle) and wild type primate TRIM5α (bottom).

Mentions: The RT-PCR product from animal 173-02 was cloned and multiple, insert-containing clones were sequenced. For every clone analyzed, the insert sequence was predicted to encode a TRIM5-CypA fusion protein. Furthermore, in every case, the demarcation between TRIM5 and CypA sequences occurred precisely at a known mRNA splice site, indicating that hybrid transcripts were not artifacts generated by RT-PCR. Two types of transcripts were detected. In some clones (n = 6), the hybrid transcript was formed by splicing from the 3′ terminus of TRIM5 exon 4 to a CypA ORF. The 5′ splice site of exon 4 follows the first nucleotide in a codon, and splicing to the CypA 3′ss from exon 4 results in a frame shift relative to the CypA sequence and a stop codon soon after the splice junction. As a result, the predicted protein product of these transcripts is almost identical to the TRIM5 ε-isoform, except for the addition of 11 C-terminal amino acids derived from the CypA insertion. The remaining clones (n = 4) were formed by mRNA splicing between the 3′ terminus of exon 6 and the CypA ORF, and resulted in a single 468 amino-acid open reading frame extending from the TRIM5 AUG initiation-codon, to a UAA stop-codon at the end of the CypA ORF (Figure 3). These results are consistent with a mechanism whereby the G-to-T substitution in the intron-6 3′ss suppresses splicing to exon 7 and promotes alternative splicing to a downstream CypA coding sequence.


Evolution of a TRIM5-CypA splice isoform in old world monkeys.

Newman RM, Hall L, Kirmaier A, Pozzi LA, Pery E, Farzan M, O'Neil SP, Johnson W - PLoS Pathog. (2008)

A TRIM5-CypA transcript of macaques is generated by alternative splicing.A. Messenger RNA sequence spanning the junction between the TRIM5 exon-6 and CypA sequences. B. Predicted amino-acid sequence of rhesus TRIM5-CypA aligned with pig-tailed macaque TRIM5-CypA and owl monkey TRIMCyp. Residues that differ from rhesus TRIM5-CypA are highlighted in blue. The region encoded by exon-7 is boxed in red; as the result of alternative splicing, this sequence is present in owl monkey TRIMCyp, but is missing from old world monkey TRIM5-CypA. C. Cartoon depicting predicted protein sequences of the old world monkey TRIM5-CypA protein (top), the owl monkey TRIM-Cyp protein (middle) and wild type primate TRIM5α (bottom).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279257&req=5

ppat-1000003-g003: A TRIM5-CypA transcript of macaques is generated by alternative splicing.A. Messenger RNA sequence spanning the junction between the TRIM5 exon-6 and CypA sequences. B. Predicted amino-acid sequence of rhesus TRIM5-CypA aligned with pig-tailed macaque TRIM5-CypA and owl monkey TRIMCyp. Residues that differ from rhesus TRIM5-CypA are highlighted in blue. The region encoded by exon-7 is boxed in red; as the result of alternative splicing, this sequence is present in owl monkey TRIMCyp, but is missing from old world monkey TRIM5-CypA. C. Cartoon depicting predicted protein sequences of the old world monkey TRIM5-CypA protein (top), the owl monkey TRIM-Cyp protein (middle) and wild type primate TRIM5α (bottom).
Mentions: The RT-PCR product from animal 173-02 was cloned and multiple, insert-containing clones were sequenced. For every clone analyzed, the insert sequence was predicted to encode a TRIM5-CypA fusion protein. Furthermore, in every case, the demarcation between TRIM5 and CypA sequences occurred precisely at a known mRNA splice site, indicating that hybrid transcripts were not artifacts generated by RT-PCR. Two types of transcripts were detected. In some clones (n = 6), the hybrid transcript was formed by splicing from the 3′ terminus of TRIM5 exon 4 to a CypA ORF. The 5′ splice site of exon 4 follows the first nucleotide in a codon, and splicing to the CypA 3′ss from exon 4 results in a frame shift relative to the CypA sequence and a stop codon soon after the splice junction. As a result, the predicted protein product of these transcripts is almost identical to the TRIM5 ε-isoform, except for the addition of 11 C-terminal amino acids derived from the CypA insertion. The remaining clones (n = 4) were formed by mRNA splicing between the 3′ terminus of exon 6 and the CypA ORF, and resulted in a single 468 amino-acid open reading frame extending from the TRIM5 AUG initiation-codon, to a UAA stop-codon at the end of the CypA ORF (Figure 3). These results are consistent with a mechanism whereby the G-to-T substitution in the intron-6 3′ss suppresses splicing to exon 7 and promotes alternative splicing to a downstream CypA coding sequence.

Bottom Line: The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform.HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A.Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts, United States of America.

ABSTRACT
The TRIM family proteins share a conserved arrangement of three adjacent domains, an N-terminal RING domain, followed by one or two B-boxes and a coiled-coil, which constitutes the tripartite-motif for which the family is named. However, the C-termini of TRIM proteins vary, and include at least nine evolutionarily distinct, unrelated protein domains. Antiviral restriction factor TRIM5alpha has a C-terminal B30.2/SPRY domain, which is the major determinant of viral target specificity. Here, we describe the evolution of a cyclophilin-A encoding exon downstream of the TRIM5 locus of Asian macaques. Alternative splicing gives rise to chimeric transcripts encoding the TRIM motif fused to a C-terminal CypA domain (TRIM5-CypA). We detected TRIM5-CypA chimeric transcripts in primary lymphocytes from two macaque species. These were derived in part from a CypA pseudogene in the TRIM5 locus, which is distinct from the previously described CypA insertion in TRIM5 of owl monkeys. The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform. All pig-tailed macaques (M. nemestrina) screened were homozygous for the CypA insertion. In contrast, the CypA-containing allele was present in 17% (17/101) of rhesus macaques (M. mulatta). The block to HIV-1 infection in lymphocytes from animals bearing the TRIM5-CypA allele was weaker than that in cells from wild type animals. HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A. Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1. Despite its distinct evolutionary origin, Macaca TRIM5-CypA has a similar domain arrangement and shares approximately 80% amino-acid identity with the TRIMCyp protein of owl monkeys. The independent appearance of TRIM5-CypA chimeras in two primate lineages constitutes a remarkable example of convergent evolution. Based on the presence of the CypA insertion in separate macaque lineages, and its absence from sooty mangabeys, we estimate that the Macaca TRIM5-CypA variant appeared 5-10 million years ago in a common ancestor of the Asian macaques. Whether the formation of novel genes through alternative splicing has played a wider role in the evolution of the TRIM family remains to be investigated.

Show MeSH
Related in: MedlinePlus