NF1 mRNA isoform expression in PC12 cells: modulation by extrinsic factors.

The rat neurofibromatosis type I (NF1) gene expresses several transcript isoforms which differ by the alternative splicing of exons 23a and 23b in the region encoding the GTPase-activating protein-related domain. The significance of this alternative splicing event is unclear and the factors which influence isoform expression are largely unknown. Here we show that a variety of factors can modulate the expression of these isoforms in PC12 cells. Nerve growth factor and dexamethasone lead to an increase in the type I isoform concurrent with a decrease in cellular proliferation. Upregulation of the type I isoform by dexamethasone occurs in an RNA synthesis-dependent manner. Cycloheximide treatment leads to the detection of an additional species identified as the murine type III transcript. These results suggest that the NF1 alternative splicing event can respond to environmental cues. The changes in the type of NF1 transcript expressed may be important in the normal physiological regulation of neurofibromin and may modulate its role in differentiation and proliferation.

The neurofibromatosis type I messenger RNA undergoes base-modification RNA editing.

A functional mooring sequence, known to be required for apolipoprotein B (apoB) mRNA editing, exists in the mRNA encoding the neurofibromatosis type I (NF1) tumor suppressor. Editing of NF1 mRNA modifies cytidine in an arginine codon (CGA) at nucleotide 2914 to a uridine (UGA), creating an in frame translation stop codon. NF1 editing occurs in normal tissue but was several-fold higher in tumors. In vitro editing and transfection assays demonstrated that apoB and NF1 RNA editing will take place in both neural tumor and hepatoma cells. Unlike apoB, NF1 editing did not demonstrate dependence on rate-limiting quantities of APOBEC-1 (the apoB editing catalytic subunit) suggesting that different trans-acting factors may be involved in the two editing processes.

Genetic and epigenetic mechanisms in the pathogenesis of neurofibromatosis type I.

Neurofibromatosis type I (NF1) is a common genetic disease which leads to a variety of clinical features affecting cells of neural crest origin. In the period since the NF1 gene was isolated in 1991, our understanding of the genetics of NF1 has increased remarkably. One of the most striking aspects of NF1 genetics is its complexity, both in terms of gene organization and expression. The gene is large and, when mutated, gives rise to diverse manifestations. A growing body of data suggests that mutations in the NF1 gene alone may not be responsible for all of the features of this disease. Epigenetic mechanisms, those which affect the NF1 transcript, play a role in the normal expression of the NF1 gene. Therefore, aberrations in those epigenetic processes are most likely pathogenic. Herein we summarize salient aspects of the vast body of NF1 literature and provide some insights into the myriad of regulatory mechanisms that may go awry in the genesis of this common but complex disease.

Effect of the kinase inhibitor 2-aminopurine on the cell cycle stage-dependent phosphorylation state of the retinoblastoma protein.

We investigated the effect of 2-aminopurine (2-AP) on the cell cycle stage-dependent phosphorylation state of the retinoblastoma growth suppressor protein (pRB). Our results suggest that 2-AP does not affect dephosphorylation of pRB scheduled during metaphase/anaphase of mitosis, even though 2-AP-treated cells never enter metaphase or anaphase. Phosphorylation of pRB, which is important for G1 exit, appeared to be impeded by 2-AP late during G1, coincident with a slowing of G1 cell progression into S phase. The significance of these data with regard to cell cycle regulation is discussed.