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HuR-regulated mRNAs associated with nuclear hnRNP A1-RNP complexes.

Papadodima O, Chatziioannou A, Patrinou-Georgoula M, Kolisis FN, Pletsa V, Guialis A - Int J Mol Sci (2013)

Bottom Line: The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes.Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs.The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicating their concerted function in mRNA metabolism.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Research and Biotechnology, Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Avenue, Athens 11635, Greece. vpletsa@eie.gr.

ABSTRACT
Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicating their concerted function in mRNA metabolism.

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Detection of pre-mRNAs of Tceal5, Gbp2 and Ccnh in hnRNP A1 IPs. (a) RT-PCR using primers specific for the unspliced transcripts were used to detect the pre-mRNA forms of the tested genes, as identified both in the total nuclear RNA and in the IPs of HuR WT and HuR KO cells. Control reactions in the absence of reverse transcriptase, to exclude the amplification of contaminated DNA, are also shown; (b) Schematic representation of the Tceal5 gene organization, showing the position of the primers used to amplify the Tceal5 pre-mRNA. RT-qPCR verification, using two different pairs of primers, of the Tceal5 pre-mRNA in IPs and total nuclear RNA in both cell types (in contrast to the spliced transcript detected only in HuR KO cells; Figure 3d).
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f4-ijms-14-20256: Detection of pre-mRNAs of Tceal5, Gbp2 and Ccnh in hnRNP A1 IPs. (a) RT-PCR using primers specific for the unspliced transcripts were used to detect the pre-mRNA forms of the tested genes, as identified both in the total nuclear RNA and in the IPs of HuR WT and HuR KO cells. Control reactions in the absence of reverse transcriptase, to exclude the amplification of contaminated DNA, are also shown; (b) Schematic representation of the Tceal5 gene organization, showing the position of the primers used to amplify the Tceal5 pre-mRNA. RT-qPCR verification, using two different pairs of primers, of the Tceal5 pre-mRNA in IPs and total nuclear RNA in both cell types (in contrast to the spliced transcript detected only in HuR KO cells; Figure 3d).

Mentions: The early loading of the hnRNP proteins on the nascent RNA pol II transcripts of pre-spliced mRNAs has been well documented [1,2]. This is also the case of HuR that binds pre-mRNA forms [37] and, as inferred from our previous findings, has the ability to associate (in addition to RNA processed low-salt-released nuclear hnRNPs) with the nuclear matrix-bound and chromatin-released hnRNP A1-RNP complexes; the latter anticipated to represent more nascent transcripts [31]. We, thus, searched for the presence, within the immunoselected nuclear hnRNP A1-RNPs of the pre-mRNA forms corresponding to three of the validated mRNA transcripts (Tceal5, Ccnh and Gbp2). RT-qPCR assays were repeated on the nuclear RNA population and the immunoprecipitated RNAs of HuR WT and HuR KO cells by designing pairs of RNA primers (File S1) so that one primer per pair aligned within an exon and the other in the neighboring intron to amplify the unspliced, pre-mRNA product. As seen in Figure 4a, the pre-mRNA form of the tested genes was clearly identified, in the RNA pool and the IP pellets of both cell types. Control reactions without reverse transcriptase were performed to exclude the amplification of contaminated DNA. This finding, therefore, indicated the integrity and stability of our immunopurified nuclear hnRNP A1-RNP complexes by their ability to sustain binding of both pre- and spliced mRNAs.


HuR-regulated mRNAs associated with nuclear hnRNP A1-RNP complexes.

Papadodima O, Chatziioannou A, Patrinou-Georgoula M, Kolisis FN, Pletsa V, Guialis A - Int J Mol Sci (2013)

Detection of pre-mRNAs of Tceal5, Gbp2 and Ccnh in hnRNP A1 IPs. (a) RT-PCR using primers specific for the unspliced transcripts were used to detect the pre-mRNA forms of the tested genes, as identified both in the total nuclear RNA and in the IPs of HuR WT and HuR KO cells. Control reactions in the absence of reverse transcriptase, to exclude the amplification of contaminated DNA, are also shown; (b) Schematic representation of the Tceal5 gene organization, showing the position of the primers used to amplify the Tceal5 pre-mRNA. RT-qPCR verification, using two different pairs of primers, of the Tceal5 pre-mRNA in IPs and total nuclear RNA in both cell types (in contrast to the spliced transcript detected only in HuR KO cells; Figure 3d).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3821614&req=5

f4-ijms-14-20256: Detection of pre-mRNAs of Tceal5, Gbp2 and Ccnh in hnRNP A1 IPs. (a) RT-PCR using primers specific for the unspliced transcripts were used to detect the pre-mRNA forms of the tested genes, as identified both in the total nuclear RNA and in the IPs of HuR WT and HuR KO cells. Control reactions in the absence of reverse transcriptase, to exclude the amplification of contaminated DNA, are also shown; (b) Schematic representation of the Tceal5 gene organization, showing the position of the primers used to amplify the Tceal5 pre-mRNA. RT-qPCR verification, using two different pairs of primers, of the Tceal5 pre-mRNA in IPs and total nuclear RNA in both cell types (in contrast to the spliced transcript detected only in HuR KO cells; Figure 3d).
Mentions: The early loading of the hnRNP proteins on the nascent RNA pol II transcripts of pre-spliced mRNAs has been well documented [1,2]. This is also the case of HuR that binds pre-mRNA forms [37] and, as inferred from our previous findings, has the ability to associate (in addition to RNA processed low-salt-released nuclear hnRNPs) with the nuclear matrix-bound and chromatin-released hnRNP A1-RNP complexes; the latter anticipated to represent more nascent transcripts [31]. We, thus, searched for the presence, within the immunoselected nuclear hnRNP A1-RNPs of the pre-mRNA forms corresponding to three of the validated mRNA transcripts (Tceal5, Ccnh and Gbp2). RT-qPCR assays were repeated on the nuclear RNA population and the immunoprecipitated RNAs of HuR WT and HuR KO cells by designing pairs of RNA primers (File S1) so that one primer per pair aligned within an exon and the other in the neighboring intron to amplify the unspliced, pre-mRNA product. As seen in Figure 4a, the pre-mRNA form of the tested genes was clearly identified, in the RNA pool and the IP pellets of both cell types. Control reactions without reverse transcriptase were performed to exclude the amplification of contaminated DNA. This finding, therefore, indicated the integrity and stability of our immunopurified nuclear hnRNP A1-RNP complexes by their ability to sustain binding of both pre- and spliced mRNAs.

Bottom Line: The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes.Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs.The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicating their concerted function in mRNA metabolism.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Research and Biotechnology, Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Avenue, Athens 11635, Greece. vpletsa@eie.gr.

ABSTRACT
Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicating their concerted function in mRNA metabolism.

Show MeSH
Related in: MedlinePlus