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Interplay between transcriptional and post-transcriptional regulation of Cyp2a5 expression
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
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2003 (English)In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1873-2968, Vol. 65, no 10, 1653-1661 p.Article in journal (Refereed) Published
Abstract [en]

The cytochrome P450 (Cyp) 2a5 gene can be upregulated transcriptionally or by mRNA stabilization. The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 interacting with the CYP2A5 mRNA has been shown to be a key post-transcriptional regulator of the Cyp2a5 gene. The aim of this study was to investigate if the transcriptional and post-transcriptional steps of Cyp2a5 expression are linked. This was done by modifying the transcription rate with transcriptional inducers (phenobarbital and cyclic AMP) and inhibitors (actinomycin D and 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole) and analyzing the effects upon post-transcriptional events. We found that inhibition of transcription led to relocalization of hnRNP A1 from the nucleus to the cytoplasm, to its strongly increased binding to the cytoplasmic CYP2A5 mRNA and to CYP2A5 mRNA stabilization. In contrast, stimulated transcription resulted in increased binding of nuclear hnRNP A1 to the Cyp2a5 promoter, and overexpression of hnRNP A1 led to stimulated transcription of a Cyp2a5 promoter-driven luciferase recombinant. This strongly suggests that the transcriptional and post-transcriptional stages of Cyp2a5 expression are interrelated and that the nucleocytoplasmic shuttling hnRNP A1 may coordinate these different steps.

Place, publisher, year, edition, pages
2003. Vol. 65, no 10, 1653-1661 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-90386DOI: 10.1016/S0006-2952(03)00118-7PubMedID: 12754101OAI: oai:DiVA.org:uu-90386DiVA: diva2:162719
Available from: 2003-04-23 Created: 2003-04-23 Last updated: 2013-05-03Bibliographically approved
In thesis
1. The Multifunctional HnRNP A1 Protein in the Regulation of the Cyp2a5 Gene: Connecting Transcriptional and Posttranscriptional Processes
Open this publication in new window or tab >>The Multifunctional HnRNP A1 Protein in the Regulation of the Cyp2a5 Gene: Connecting Transcriptional and Posttranscriptional Processes
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The mouse xenobiotic-inducible Cyp2a5 gene is both transcriptionally and posttranscriptionally regulated. One of the most potent Cyp2a5 inducers, the hepatotoxin pyrazole, increases the CYP2A5 mRNA half-life. The induction is accomplished through the interaction of a pyrazole-inducible protein with a 71 nt long, putative hairpin-loop region in the 3' UTR of the CYP2A5 mRNA.

The aims of this thesis have been to identify the pyrazole-inducible protein, to investigate its role in the Cyp2a5 expression and the significance of the 71 nt hairpin-loop region for the Cyp2a5 expression, and to examine a possible coupling between transcriptional and posttranscriptional processes in Cyp2a5 expression.

The pyrazole-inducible protein was identified as the heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Studies performed in mouse primary hepatocytes overexpressing hnRNP A1, and in mouse erythroleukemia derived cells lacking hnRNP A1, revealed that the 71 nt region in the 3' UTR of the CYP2A5 mRNA is essential for Cyp2a5 expression.

The hnRNP A1 is a multifunctional nucleocytoplasmic shuttling protein, with the ability to bind both RNA and DNA. These properties make it an interesting candidate mediating a coupling between nuclear and cytoplasmic gene regulatory events, which was investigated for the Cyp2a5. In conditions of cellular stress hnRNP A1 translocates from the nucleus to the cytoplasm. The accumulation of cytoplasmic hnRNP A1 after RNA polymerase II transcription inhibition, resulted in an increased binding of hnRNP A1 to the CYP2A5 mRNA, parallel with a stabilization of the CYP2A5 mRNA.

Treating primary mouse hepatocytes with phenobarbital (PB), a Cyp2a5 transcriptional inducer, resulted in a mainly nuclear localization of the hnRNP A1. Electrophoretic mobility shift assays with nuclear extracts from control or PB-treated mice, revealed that hnRNP A1 interacts with two regions in the Cyp2a5 proximal promoter, and that the interaction to one of the regions was stimulated by PB treatment.

In conclusion, the change in hnRNP A1 subcellular localization after transcriptional inhibition or activation, together with the effects on the interaction of hnRNP A1 with the CYP2A5 mRNA and Cyp2a5 promoter, suggest that hnRNP A1 could couple the nuclear and cytoplasmic events of the Cyp2a5 expression.

The presented studies are the first showing involvement of an hnRNP protein in the regulation of a Cyp gene. Moreover, it is the first time an interconnected transcriptional and posttranscriptional regulation has been suggested for a member of the Cyp gene family.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 53 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 0282-7484 ; 289
Keyword
Pharmaceutical biochemistry, Cyp2a5, DNA-protein interaction, gene regulation, hnRNP A1, nucleocytoplasmic shuttling, RNA-protein interaction, RNA stabilization, posttranscriptional, transcriptional, Farmaceutisk biokemi
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-3412 (URN)91-554-5603-0 (ISBN)
Public defence
2003-05-17, C4:305, Uppsala biomedicinska centrum (BMC), Uppsala, 10:15
Opponent
Supervisors
Available from: 2003-04-23 Created: 2003-04-23Bibliographically approved
2. The role of hnRNP A1 and hnRNP C1/C2 in the regulation of the stress responsive genes Cyp2a5/2A6 and p53.
Open this publication in new window or tab >>The role of hnRNP A1 and hnRNP C1/C2 in the regulation of the stress responsive genes Cyp2a5/2A6 and p53.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The family of proteins known as heterogeneous nuclear ribonucleoproteins (hnRNPs) is large and diverse. Often, one and the same hnRNP will perform multiple cellular functions, leading to their description as “multifunctional proteins”. The two hnRNPs known as hnRNP A1 and hnRNP C1/C2 are multifunctional proteins found to affect the transcription, splicing, stability, and translation of specific genes’ mRNA. They are implicated in carcinogenesis, apoptosis, and DNA damage response mechanisms.

The aims of this thesis were to study the hnRNP A1 and hnRNP C1/C2 dependent regulation of two highly stress responsive genes, the tumor suppressor p53 and the cytochrome P450 enzyme Cyp2a5/CYP2A6. We identified hnRNP C1/C2 as a DNA damage induced binding protein towards the coding region of p53 mRNA, and found that while a specific cis binding site appears to have a positive function in p53 expression, interaction of hnRNP C1/C2 with this site represses the expression. The data suggest that two distinct molecular mechanisms exist for the down-regulation of p53 by hnRNP C1/C2. One mechanism, active during transcriptional stress, is dependent upon the aforementioned site, and the other, independent. We discuss how hnRNP C1/C2 dependent repression of p53 may play a role in apoptosis.

The data presented here further suggest that the transcriptional and post-transcriptional processes controlling the expression of the murine Cyp2a5 gene are linked via hnRNP A1, by performing functions in the nucleus as a transcription factor, or in the cytoplasmic compartment as a trans factor bound to the 3’UTR of the mRNA as needed. Our studies of the human ortholog of this gene, CYP2A6, suggest that this gene is regulated post-transcriptionally in a manner similar to that of its murine counterpart, via changes in mRNA stability and interaction of hnRNP A1 with its 3’ UTR.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 90 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 74
Keyword
Molecular biology, hnRNP, Tumor Suppressor Protein p53, CYP2A5, CYP2A6, Apoptosis, hnRNP C Proteins, hnRNP protein A1, Cancer, Molekylärbiologi
Identifiers
urn:nbn:se:uu:diva-8722 (URN)978-91-554-7198-9 (ISBN)
Public defence
2008-05-23, BMC/C2:301, Biomedicinsk Centrum, Husargatan 3, Uppsala, 09:15
Opponent
Supervisors
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2013-05-03Bibliographically approved

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