Finding the right RNA: Identification of cellular mRNA substrates for RNA-binding proteins

Trifillis, Panayiota; Day, Nancy A.; Kiledjian, Megerditch

doi:10.1017/s1355838299981803

Cited by 44 publications

(52 citation statements)

References 35 publications

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“…The K562 cell line was chosen for this analysis. This human erythroid cell line contains high levels of human ␣-globin and ␥-globin mRNAs which can serve as positive and negative controls, respectively, for the IP reaction; interaction of ␣-globin mRNA with ␣CP2 has been previously characterized in detail and has a defined biologic function (23), while ␥-globin mRNA lacks ␣CP binding sites (49). Native ␣CP2-RNP complexes were immunoprecipitated from K562 cytoplasmic extracts using an affinity-purified rabbit antiserum specific to the two predominant human ␣CP isoforms, ␣CP2 and ␣CP2-KL (anti-␣CP2) (6).…”

Section: Resultsmentioning

confidence: 99%

“…4E). Even though it is not present on the microarray used in these studies, we wanted to assess whether this mRNA interacts with ␣CP2 because it has been shown previously to be a binding partner of ␣CP1 (49). Figure 4E shows that coxII mRNA is robustly associated with ␣CP2-RNPs.…”

Section: Resultsmentioning

confidence: 99%

“…Five mRNAs have been identified in a screen for ␣CP1 binding targets: mitochondrial NADH dehydrogenase subunit 3, mitochondrial cytochrome c oxidase subunit II (coxII), ribosomal protein of the large subunit (L27a), palmitoylated erythrocyte membrane protein p55, manganese superoxide dismutase, and the CD81 receptor (TAPA-1) (49). The mRNAs encoding TAPA-1 and coxII were further shown to contain ␣CP1 binding sites in the 3Ј UTRs by in vitro binding assays.…”

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confidence: 99%

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Identification of mRNAs Associated with αCP2-Containing RNP Complexes

Waggoner

Liebhaber

2003

Molecular and Cellular Biology

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Posttranscriptional controls in higher eukaryotes are central to cell differentiation and developmental programs. These controls reflect sequence-specific interactions of mRNAs with one or more RNA binding proteins. The ␣-globin poly(C) binding proteins (␣CPs) comprise a highly abundant subset of K homology (KH) domain RNA binding proteins and have a characteristic preference for binding single-stranded C-rich motifs. ␣CPs have been implicated in translation control and stabilization of multiple cellular and viral mRNAs. To explore the full contribution of ␣CPs to cell function, we have identified a set of mRNAs that associate in vivo with the major ␣CP2 isoforms. One hundred sixty mRNA species were consistently identified in three independent analyses of ␣CP2-RNP complexes immunopurified from a human hematopoietic cell line (K562). These mRNAs could be grouped into subsets encoding cytoskeletal components, transcription factors, proto-oncogenes, and cell signaling factors. Two mRNAs were linked to ceroid lipofuscinosis, indicating a potential role for ␣CP2 in this infantile neurodegenerative disease. Surprisingly, ␣CP2 mRNA itself was represented in ␣CP2-RNP complexes, suggesting autoregulatory control of ␣CP2 expression. In vitro analyses of representative target mRNAs confirmed direct binding of ␣CP2 within their 3 untranslated regions. These data expand the list of mRNAs that associate with ␣CP2 in vivo and establish a foundation for modeling its role in coordinating pathways of posttranscriptional gene regulation.Programs of gene expression in eukaryotic cells are coordinated at multiple levels. A significant component of these controls is mediated by posttranscriptional mechanisms. Processing, transport, localization, stability, and translation of an mRNA are often controlled via sequence-specific interactions with RNA binding proteins. Most of these interactions are discovered in the course of exploring specific pathways of gene regulation. Such gene-specific discovery approaches, while highly informative, are not able to focus on the wider impact of particular RNA binding proteins on cell function. A complementary approach that can more effectively address this question would involve the identification of the full spectrum of mRNA targets for a particular RNA binding protein (46). Such an approach would set the stage for constructing an integrated view of how particular RNA binding proteins contribute to coordination of gene expression profiles (21).Limited examples of coordinate control by RNA binding proteins can be cited from the literature. An initial example is represented by the iron response element binding protein (8). This iron-binding protein can reversibly block translation of ferritin mRNA via 5Ј untranslated region (UTR) binding and reversibly stabilize transferrin receptor mRNA via 3Ј UTR binding. These distinct actions of the iron response element binding protein coordinate an integrated cellular response to intracellular iron levels. By defining the full-spectrum target mRNAs for specific...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of mRNAs Associated with αCP2-Containing RNP Complexes

Waggoner

Liebhaber

2003

Molecular and Cellular Biology

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“…Because the target mRNAs of many RBPs have not been identified and different RBPs may bind the same target http://bmbreports.org BMB reports mRNAs, GST protein was used as a control. In order to provide the natural circumstances of competitor proteins and increase the binding specificity of the protein-mRNA, the total embryo extract was chosen to enable the identification of true mRNA substrates (12). As a result, 13 target mRNAs were bound by GST-ZDnd protein (Table 1).…”

Section: Identification Of the Target Mrnas Of Zdnd Protein By Sannp mentioning

confidence: 99%

Zebrafish Dnd protein binds to 3'UTR of geminin mRNA and regulates its expression

Chen¹,

Zeng²,

Sun³

et al. 2010

BMB Reports

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“…This assay adopts copurification of unknown mRNAs bound by an RNA-binding protein and subsequently isolates the mRNAs using differential display technology [18]. We herein report the testis-specific substrate mRNAs bound by hRBMY.…”

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Identifying mRNAs Bound by Human RBMY Protein in the Testis

Zeng

Liang

Zhao

et al. 2011

Journal of Reproduction and Development

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Abstract. Rbmy gene encodes a germ-cell specific nuclear RNA-binding protein and is involved in spermatogenesis. To further investigate the specific events of spermatogenesis in which Rbmy plays a role, the target mRNAs of human RBMY protein were isolated and identified. Through the isolating specific nucleic acids associated with proteins (SNAAP) technique, we isolated twenty potential target genes of human RBMY protein from the human testis in the present study. Some of these target genes play important roles during spermatogenesis and have alternative transcripts in the testis. In this study, we focused on the human-related (never in mitosis gene a) kinase 10 (Nek10) gene, which belongs to the Nek protein kinase subfamily. Nek10 has two transcripts, and the results of RT-PCR and Electrophoretic Mobility Shift Assays (EMSA) show that hRBMY protein can only bind to transcript variant 2 of Nek10 and that hRbmy may take part in alternative splicing of Nek10. Isolation and identification of target genes of hRBMY will be helpful to further investigate the biological function of RBMY in spermatogenesis. Key words: Nek10, Rbmy gene, RNA-binding protein, SNAAP technique, Spermatogenesis (J. Reprod. Dev. 57: [107][108][109][110][111][112] 2011) permatogenesis is a complex cell differentiation process that requires highly regulated expression of lots of genes [1][2][3]. Many genes are mainly or exclusively expressed in the testis, and in most situations, further research has shown that they are only expressed in the germline cells [4]. Microdeletions at a specific region called the AZF in the long arm of Y chromosome have been described in significant proportions of oligo and azoospermic patients, suggesting that some genes located at these regions play an essential role during spermatogenesis [5]. Of all the genes located at the AZF region, Rbmy was the first of these candidate genes to be identified [6].There are many Rbm genes distributed throughout the Y chromosome, but only the genes in deletion interval AZFb give rise to detectable levels of protein [7]. According to the complete sequence of the human Y chromosome, it is now known that there are six Rbmy copies mapped in the AZFb region, but only one copy is actively transcribed [8]. Human and mouse Rbmy genes have a homologue on the human X chromosome (Rbmx) that maintains a widespread function, while Rbmy evolves a male-specific function in spermatogenesis [9]. There is another retrogene called hnRNPG-T that belongs to the same family as Rbmy and Rbmx, which is also specifically expressed in the testis. Previous study has shown that haploinsufficiency of hnRNPG-T results in abnormal sperm production in the mouse. Genetic defects resulting in reduced levels of hnRNPG-T may be a cause of male infertility in humans [10]. Although Rbmy is evolutionarily conserved in the human, mouse and marsupial [11][12][13], their expression patterns are not completely identical. The expression of mouse Rbmy is limited to spermatogonia and early spermatocytes, while human Rbmy ...

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Finding the right RNA: Identification of cellular mRNA substrates for RNA-binding proteins

Cited by 44 publications

References 35 publications

Identification of mRNAs Associated with αCP2-Containing RNP Complexes

Identification of mRNAs Associated with αCP2-Containing RNP Complexes

Zebrafish Dnd protein binds to 3'UTR of geminin mRNA and regulates its expression

Identifying mRNAs Bound by Human RBMY Protein in the Testis

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