Protein synthesis inhibitors lead to increased levels of murine type I and type III NF1 mRNA isoforms

Linda J. Metheny* and Gary R. Skuse

*Present address: Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Building 37,Room 2C08, 37 Convent Drive, MSC 4255, Bethesda, MD 20892-4255.


Neurofibromatosis type I (NF1) is a common genetic disease which predisposes to neoplasia and a number of other clinicalfeatures. The tissues most commonly affected in NF1 are derived from the embryonic neuroectoderm, suggesting that the product of the NF1 gene, neurofibromin, is important in establishing and maintaining proper morphology in these tissues. Neurofibromin is thought to exert a tumor suppressor function by facilitating the conversion of activated ras-GTP to the inactive ras-GDP form by stimulating the GTPase intrinsic to ras through its GTPase activating protein (GAP) domain (GRD).

The rat NF1 gene expresses several transcript isoforms which differ by the alternative splicing of exons 23a and 23b within the region encoding the GAP-related domain (GRD). The type I isoform lacks either exon while inclusion of the 63 base pair exon 23a gives rise to the type II transcript. This insertion decreases both the GAP activity and the microtubule binding activity of the encoded protein. The inclusion of exon 23b in the type II species results in the type III isoform. This 41 base insertion results in a frameshift which introduces a termination codon in exon 24, such that the resulting protein, if produced, would lack most of the GRD. The alternative splicing events, therefore, result in mRNAs which may encode vastly different forms of neurofibromin. Although the specific upregulation of the type I isoform has been reported under various conditions, few studies have attempted to characterize the mechanism of that upregulation and fewer yet have focused on modulation of the type III isoform expression.

We have previously shown that nerve growth factor, dexamethasone, and the protein synthesis inhibitor cycloheximide each can, to varying extents, stimulate an increase in the levels of NF1 type I mRNA in PC12 cells. While the increase elicited by cycloheximide is relatively small (only two- to threefold by 24 hours), it is fairly rapid and allows us a means of investigating the mechanism by which type I isoform levels may be increased. Additionally, we have reported that cycloheximide treatment results in the detection of the murine type III transcript which can account for up to 20% of the total NF1 mRNA detected in cycloheximide-treated PC12 cells.

Cycloheximide has been shown to exert a number of different effects on cells; first and most well-characterized is its ability to inhibit translational elongation. Additionally, cycloheximide has been demonstrated to increase the stability of a number of mRNAs, including those which are slated for nonsense-mediated decay. More recently, several studies have shown that cycloheximide has the ability to alter signal transduction cascades and stimulate the transcription of immediate early genes at concentrations below those necessary for the inhibition of translational elongation.

To gain insight into how the type I and type III NF1 isoforms are regulated, we sought to determine which of the pleiotropic effects attributed to cycloheximide may account for the type I and type III increases observed. To do this, we used other agents which inhibit protein synthesis by similar or different mechanisms (i.e. anisomycin and puromycin respectively). These agents also have differential effects on gene expression, and may help to unravel the mechanisms underlying NF1 isoform level increases. Previously, despite extensive study of the GRD region, no one has reported an exon 23b-containing mRNA isoform in humans. Since that form was only detected in PC12 cells following treatment with cycloheximide we sought to determine if the type III increase could be elicited in a similar fashion in human cells but could not detect any.

In this report, we demonstrate that protein synthesis inhibitors can induce NF1 type I isoform expression in a fashion that is independent of the ability to completely inhibit protein synthesis. This occurs not by stabilization of the type I transcript, but by stimulation of a tyrosine phosphorylation-dependent pathway. Further, this mechanism for upregulation appears to be used in a variety of species and tissue types, providing the first insight into the mechanisms responsible for differential NF1 transcript isoform expression.

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