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The Multifunctional HnRNP A1 Protein in the Regulation of the Cyp2a5 Gene: Connecting Transcriptional and Posttranscriptional Processes
Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
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. , p. 53
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 0282-7484 ; 289
Keywords [en]
Pharmaceutical biochemistry, Cyp2a5, DNA-protein interaction, gene regulation, hnRNP A1, nucleocytoplasmic shuttling, RNA-protein interaction, RNA stabilization, posttranscriptional, transcriptional
Keywords [sv]
Farmaceutisk biokemi
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-3412ISBN: 91-554-5603-0 (print)OAI: oai:DiVA.org:uu-3412DiVA, id: diva2:162721
Public defence
2003-05-17, C4:305, Uppsala biomedicinska centrum (BMC), Uppsala, 10:15
Opponent
Supervisors
Available from: 2003-04-23 Created: 2003-04-23 Last updated: 2018-01-13Bibliographically approved
List of papers
1. Heterogeneous nuclear ribonucleoprotein A1 and regulation of the xenobiotic-inducible gene Cyp2a5
Open this publication in new window or tab >>Heterogeneous nuclear ribonucleoprotein A1 and regulation of the xenobiotic-inducible gene Cyp2a5
2002 In: Molecular Pharmacology, Vol. 61, no 4, p. 795-99Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-90384 (URN)
Available from: 2003-04-23 Created: 2003-04-23Bibliographically approved
2. Interplay between hnRNP A1 and a cis-acting element in the 3' UTR of CYP2A5 mRNA is central for high expression of the gene
Open this publication in new window or tab >>Interplay between hnRNP A1 and a cis-acting element in the 3' UTR of CYP2A5 mRNA is central for high expression of the gene
2003 In: FEBS Letters, Vol. 535, p. 147-52Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-90385 (URN)
Available from: 2003-04-23 Created: 2003-04-23Bibliographically approved
3. Interplay between transcriptional and post-transcriptional regulation of Cyp2a5 expression
Open this publication in new window or tab >>Interplay between transcriptional and post-transcriptional regulation of Cyp2a5 expression
Show others...
2003 (English)In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 65, no 10, p. 1653-1661Article 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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-90386 (URN)10.1016/S0006-2952(03)00118-7 (DOI)12754101 (PubMedID)
Available from: 2003-04-23 Created: 2003-04-23 Last updated: 2017-12-14Bibliographically approved
4. Passage-dependent expression of an hnRNP A1-like protein in mouse erythroleukemia derived cells with a disrupted hnrnpa1 gene
Open this publication in new window or tab >>Passage-dependent expression of an hnRNP A1-like protein in mouse erythroleukemia derived cells with a disrupted hnrnpa1 gene
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-90387 (URN)
Available from: 2003-04-23 Created: 2003-04-23 Last updated: 2010-01-13Bibliographically approved

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