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1.
Mol Cell Proteomics ; 23(3): 100718, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38224738

RESUMO

A functional role has been ascribed to the human dihydrofolate reductase 2 (DHFR2) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivo function is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homolog, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2-specific peptides as evidence of its translation. We show definitive evidence that the DHFR2 activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, are differentially associated with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2-specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2 gene in adult cells is likely to arise at the RNA level.


Assuntos
RNA , Tetra-Hidrofolato Desidrogenase , Humanos , Linhagem Celular , Peptídeos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
2.
Methods Mol Biol ; 2324: 239-254, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34165719

RESUMO

The technical challenge in proving that a given expressed pseudogene is in fact translated into a functional protein is specificity. To circumvent this challenge, one approach is to use PCR in order to generate a series of clones that allow one to exogenously express the pseudogenic protein of interest, either native or fused to a tag, which can facilitate purification, detection, and complementation in both bacterial and mammalian cells. This approach allows an assessment of whether a putative pseudogenic protein possesses enzymatic activity, to identify its subcellular localization and to test its capacity to complement the parental homolog. An alternative approach is to detect the endogenous protein using targeted proteomics analysis and to assess the full range of endogenous RNA isoforms, in order to consider additional coding and noncoding RNA functionality.


Assuntos
Biossíntese de Proteínas , Proteômica/métodos , Pseudogenes , RNA Mensageiro/genética , Animais , Linhagem Celular , Cromatografia Líquida/métodos , Clonagem Molecular/métodos , Sondas de DNA , Escherichia coli , Vetores Genéticos/genética , Humanos , Mamíferos , Fases de Leitura Aberta/genética , Pseudogenes/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Frações Subcelulares/química , Espectrometria de Massas em Tandem/métodos
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