Identification and distribution of different mRNA variants produced by differential splicing in the human phosphodiesterase 9A gene

doi:10.1016/S0006-291X(03)00021-4

Biochemical and Biophysical Research Communications

Volume 301, Issue 3, 14 February 2003, Pages 686-692

https://doi.org/10.1016/S0006-291X(03)00021-4 Get rights and content

Abstract

The transcript population of the human gene coding for a cGMP-dependent phosphodiesterase (PDE9A) has a complex structure. There is a high level of mRNA in intestinal and prostate tissues, a low level in blood, and intermediate in other tissues. More than 20 different variants produced by differential splicing have been observed and new exons have been identified both by PCR amplification and by the analysis of available EST sequences. In all cases the transcriptional start site is the same and no differential splicing is found in the exons coding for the catalytic domain of the protein. In some cases the protein produced by splice variants is truncated. The distribution of the splice variants is not homogeneous among the different tissues studied. The human, but not the mouse, PDE9A gene appears to have a complex regulation of expression by different isoforms.

Section snippets

Materials and methods

Cell culture. HeLa cells (human cervix carcinoma cell line) were grown at 37 °C under 5% CO₂ in Dulbecco’s modified Eagle’s medium with 10% foetal bovine serum. Jurkat cells (human T cell leukemia cell line) were cultured, harvested, and resuspended as previously described [13]. Caco2 cells (human colon adenocarcinoma cell line) were grown at 37 °C under 5% CO₂ in Dulbecco’s modified Eagle’s medium with 10% foetal bovine serum, 2 mM l-glutamine, and 1% non-essential amino acids.

RNA blot and dot

Accumulation of PDE9A mRNA: RNA and dot blot analyses

In order to find an appropriate system to study the accumulation of PDE9A mRNA and its population of splice variants, total RNA of HeLa and Jurkat cells was isolated. An RNA blot was then hybridised with a ³²P-radiolabelled oligonucleotide (nt 541–587) probe. A band was found around 1.9 kb (Fig. 1), in accordance with the expected size of the PDE9A mRNA. The band often appears broad or as a smear, which may indicate the existence of several mRNAs for the same gene.

The accumulation pattern of the

Acknowledgements

We thank David Piñeyro and Dr. Mònica Torras for continuous interest and help. We also thank Dr. Arsenio Nueda from Laboratorios Almirall-Prodesfarma S.A. (Barcelona) and Dr. Ferran Azorı́n (IBMB-CSIC, Barcelona) for providing HeLa, Jurkat, and Caco2 cells. This work was supported by a grant from the Spanish Ministerio de Ciencia y Tecnologı́a, (Proyectos FEDER) and it has been carried out within the framework of the Centre de Referència de Biotecnologia de la Generalitat de Catalunya.

References (31)

S.H. Soderling et al.
Regulation of cAMP and cGMP signalling: new phosphodiesterases and new functions
Curr. Opin. Cell Biol.
(2000)
J.A. Beavo et al.
Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors
Trends Pharmacol. Sci.
(1990)
D.A. Fisher et al.
Isolation and characterisation of PDE9A, a novel human cGMP-specific phosphodiesterase
J. Biol. Chem.
(1998)
S.H. Soderling et al.
Identification and characterisation of a novel family of cyclic nucleotide phosphodiesterase
J. Biol. Chem.
(1998)
E. Degerman et al.
Structure, localisation, and regulation of cGMP-inhibited phosphodiesterase (PDE3)
J. Biol. Chem.
(1997)
X. Miró et al.
Phosphodiesterases 4D and 7A splice variants in the response of HUVEC cells to TNF-α
Biochem. Biophys. Res. Commun.
(2000)
M. Pompeiano et al.
Distribution of the serotonin 5-HT2 receptor family of mRNAs: comparison between 5-HT2A and 5-HT2C receptors
Mol. Brain Res.
(1994)
M.T. Vilaro et al.
Muscarinic M2 receptor mRNA expression and receptor binding in cholinergic and non-cholinergic cells in the rat brain: a correlative study using in situ hybridisation histochemistry and receptor autoradiography
Neuroscience
(1992)
K. Yuasa et al.
Isolation and characterization of two novel phosphodiesterase PDE11A variants showing unique structure and tissue-specific expression
J. Biol. Chem.
(2000)
M.J. Perry et al.
Chemotherapeutic potential of phosphodiesterase inhibitors
Curr. Opin. Chem. Biol.
(1998)

M. Sullivan et al.

Genomic organisation of the human cyclic AMP-specific phosphodiesterase PDE4C gene and its chromosomal localisation to 19q13.1, between RAB3A and JUND

Cell Signal.

(1999)

M. Fidock et al.

Isolation and differential tissue distribution of two human cDNAs encoding PDE1 splice variants

Cell Signal.

(2002)

P. Wang et al.

Human phosphodiesterase 8A splice variants: cloning, gene organization, and tissue distribution

Gene

(2001)

M. Hayashi et al.

Genomic organization, chromosomal localization, and alternative splicing of the human phosphodiesterase 8B gene

Biochem. Biophys. Res. Commun.

(2002)

S.H. Francis et al.

Cyclic nucleotide phosphodiesterases: relating structure and function

Prog. Nucleic Acid Res. Mol. Biol.

(2001)

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Identification and distribution of different mRNA variants produced by differential splicing in the human phosphodiesterase 9A gene

Abstract

Section snippets

Materials and methods

Accumulation of PDE9A mRNA: RNA and dot blot analyses

Acknowledgements

Curr. Opin. Cell Biol.

Trends Pharmacol. Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Mol. Brain Res.

Neuroscience

J. Biol. Chem.

Curr. Opin. Chem. Biol.

Cell Signal.

Cell Signal.

Gene

Biochem. Biophys. Res. Commun.

Cyclic nucleotide phosphodiesterases: relating structure and function

Prog. Nucleic Acid Res. Mol. Biol.