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. 2021:2324:239-254.
doi: 10.1007/978-1-0716-1503-4_15.

Methods to Study Translated Pseudogenes: Recombinant Expression and Complementation, Targeted Proteomics, and RNA Profiling

Anne Parle-McDermott  1   2 , Niamh Bookey  3   4 , Paula Meleady  4 , Paola Drago  3   4
Affiliations

Methods to Study Translated Pseudogenes: Recombinant Expression and Complementation, Targeted Proteomics, and RNA Profiling

Anne Parle-McDermott et al. Methods Mol Biol. 2021.

Abstract

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.

Keywords: Cloning; DHFR; Expression proteomics; Pseudogene; RNA profiling.

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References

    1. Ma D, Baruch D, Shu Y, Yuan K, Sun Z, Ma K, Hoang T, Fu W, Min L, Lan Z-S, Wang F, Mull L, He W-W (2012) Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology. BMC Biotechnol 12(1):88. https://doi.org/10.1186/1472-6750-12-88 - DOI - PubMed - PMC
    1. Zhang Z, Harrison PM, Liu Y, Gerstein M (2003) Millions of years of evolution preserved: a comprehensive catalog of the processed pseudogenes in the human genome. Genome Res 13(12):2541–2558. https://doi.org/10.1101/gr.1429003 - DOI - PubMed - PMC
    1. McEntee G, Minguzzi S, O’Brien K, Larbi NB, Loscher C, Ó’Fágáin C, Parle-McDermott A (2011) The former annotated human pseudogene dihydrofolate reductase-like 1 (DHFRL1) is expressed and functional. Proc Natl Acad Sci 108(37):15157–15162. https://doi.org/10.1073/pnas.1103605108 - DOI - PubMed - PMC
    1. Brosius J (1999) RNAs from all categories generate retrosequences that may be exapted as novel genes or regulatory elements. Gene 238(1):115–134. https://doi.org/10.1016/s0378-1119(99)00227-9 - DOI - PubMed
    1. Li C, Wen A, Shen B, Lu J, Huang Y, Chang Y (2011) FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method. BMC Biotechnol 11(1):92. https://doi.org/10.1186/1472-6750-11-92 - DOI - PubMed - PMC

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