The c-myc mRNA Coding Region Determinant-Binding Protein
September 1, 2000
The c-myc mRNA Coding Region Determinant-Binding Protein
By Jeffrey Ross, MD
The coding region determinant-binding protein (crd-bp) was identified in our lab as a protein that binds, with high specificity, to the coding region of the mRNA encoding c-Myc, a nuclear phosphoprotein and oncogene product. Homologues of the CRD-BP capable of binding to other mRNAs also have been found by other laboratories. The significance of the CRD-BP, with respect to c-myc mRNA binding stems from the central role of c-Myc protein in cell biology and tumorigenesis.
Background
c-Myc is a helix-loop-helix/leucine zipper (HLH/LZ) type transcription factor whose expression plays a major role in cell proliferation, differentiation, and neoplastic transformation.1 Regulated c-myc expression apparently is essential for determining whether a cell will differentiate. For example, c-myc mRNA is down-regulated when erythroid cell lines are induced to differentiate following exposure to dimethylsulfoxide. However, if the cells constitutively express c-myc from a transgene that is not down-regulated, the cells fail to differentiate. Contrary to that, blocking c-myc expression prevents entry into S phase and promotes terminal differentiation. c-myc expression also is controlled during cell growth transitions, being up-regulated 50-fold or more as quiescent cells enter the cell cycle.
Two additional observations support an active, causal role for c-Myc as a regulator of cell differentiation and replication. First, constitutive expression of c-Myc can force cells to enter S phase or to undergo apoptosis under conditions of serum starvation. Second, abnormal c-Myc regulation resulting from gene amplification, proviral insertion, or chromosomal translocation induces tumors in animals. c-Myc expression is crucial for embryonic development in rodents, because homozygous knockout of the c-MYC gene in transgenic mice causes them to die in utero.2 c-MYC also is one of the "early response" genes that is up-regulated when quiescent adult hepatocytes are induced to replicate following partial hepatectomy (surgical excision of 70% of the liver mass). c-myc mRNA is barely detectable in adult liver but is up-regulated and easily detectable within two hours post-hepatectomy.3
The abundance of c-Myc protein is influenced, to a large extent, by changes in c-myc mRNA stability. The half-life of c-myc mRNA usually is 15-30 minutes but can be two hours or more in some cells under certain conditions. For example, the c-myc gene is active and c-myc mRNA is relatively stable in fetal rodent hepatocytes. As a result, fetal hepatocytes contain abundant c-myc mRNA. The c-myc gene is also actively transcribed in adult hepatocyes, but the mRNA is quite unstable, so that adult hepatocytes contain little or no c-myc mRNA.3 Two cis-acting sequence elements affect the half-life of c-myc mRNA: an AU-rich element (AURE) in the 3’-untranslated region (3’-UTR); and a ~250 nucleotide coding region instability determinant (CRD).4 The CRD encodes part of the HLH/LZ domain near the C-terminus of the protein.
Four observations show that the CRD of c-myc mRNA functions independently of the AURE to affect mRNA expression. First, c-myc mRNA, lacking its CRD, is more stable than wild-type c-myc mRNA.4 Second, the CRD is required for the post-transcriptional down-regulation of c-myc mRNA that occurs when cultured myoblasts fuse to form myotubes.5 Third, the c-myc CRD functions as an mRNA-destabilizing element when fused in frame within the coding region of b-globin mRNA, which is normally very stable.4 In other words, placing the CRD-BP from c-myc mRNA within the coding region of b-globin mRNA causes the resulting chimeric mRNA (part globin, part c-myc) to become less stable than b-globin mRNA itself. Fourth, the c-myc CRD is required for up- and down-regulating c-myc mRNA abundance post-transcriptionally in the livers of transgenic mice undergoing liver regeneration following partial hepatectomy.3
CRD-BP and c-myc Expression
We discovered the CRD-BP and investigated how it affects c-myc expression in experiments by exploiting a cell-free mRNA decay system. The standard reaction mixture includes polysomes isolated from cultured mammalian cells. The polysomes contain both the substrates (mRNAs) for decay and at least some of the enzymes and co-factors that affect mRNA stability. Polysomes are incubated in an appropriate buffer, and the decay rates of endogenous, polysomal mRNAs such as c-myc are monitored by hybridization. This in vitro assay reflects many aspects of mRNA decay in intact cells.
For example, mRNAs such as c-myc that are unstable in cells are also unstable in vitro, while mRNAs such as globin that are stable in cells are stable in vitro.6 In standard mRNA decay reactions, polysome-associated c-myc mRNA is degraded rapidly in a 3’ to 5’ direction, perhaps by an exonuclease. An alternative endonucleolytic decay pathway is activated when the reactions are supplemented with a 180 nucleotide sense strand competitor RNA corresponding to part of the c-myc CRD. This RNA induces endonucleolytic cleavage within the c-myc CRD, thereby destabilizing c-myc mRNA 4- to 8-fold.7 In other words, although the mRNA is normally degraded very rapidly under standard conditions, it is even less stable when the competitor RNA is added. This destabilization effect is c-myc-specific. Other competitor RNAs do not destabilize c-myc mRNA, and c-myc CRD competitor RNA does not destabilize other mRNAs tested.
A Model for CRD-BP Activity
Based on these observations, we suggested the model shown in the Figure.
The model shows: 1) the c-myc CRD is normally susceptible to attack by a ribosome-associated endoribonuclease (RNase); 2) however, a protein (the CRD-BP) can bind to the CRD and shield the CRD from the RNase; and 3) in vitro, when we add competitor CRD RNA to the reactions, the competitor titrates the protein off c-myc mRNA, leaving the CRD unprotected and open to attack by the RNase. Consistent with this model, we detected a protein that binds in vitro to c-myc CRD 32P-RNA.7 We also have partially purified a ribosome-associated endoribonuclease whose properties match those of the RNase that degrades c-myc mRNA.8 It has not been proven, however, that this RNase is the same enzyme responsible for attacking the c-myc CRD when the CRD-BP is not bound.
Identifying the CRD-BP Gene
The c-myc CRD-BP has been purified to homogeneity.9 Its cDNA has been cloned and sequenced, and the mouse CRD-BP gene has been characterized in some detail.9-11 Sequencing the cDNA revealed that the CRD-BP is orthologous to several proteins that bind to other mRNAs, including chicken ß-actin and human insulin-like growth factor II.12,13 It also is related to a human protein overexpressed in pancreatic cancer, a human hepatocellular carcinoma autoantigen, and several developmentally regulated Xenopus proteins.14-18 These proteins are discussed further later in this section.
The CRD-BP primarily is a cytoplasmic protein that co-sediments with polysomes, which is consistent with its proposed role in shielding polysomal c-myc mRNA from endonucleolytic attack. It contains 577 amino acids and is a member of a family of RNA-binding proteins containing several motifs that are characteristic of many RNA-binding proteins. The most prominent such motif is the so-called KH (K-homology) domain, four of which are present in the protein. KH domains are found in a wide variety of RNA-binding proteins with different functions. The finding of well-characterized RNA-binding domains encoded by the cDNA, coupled with the fact that recombinant CRD-BP synthesized from the cDNA binds to c-myc CRD RNA, provides strong evidence that the cDNA in fact encodes the CRD-BP. The CRD-BP gene has 15 exons and 14 introns, is single-copy, and is located on chromosome 11 in mice and 17 in humans, close to HER-2/neu.11
An intriguing feature of the CRD-BP is that different laboratories have identified it based on very different assays. Several laboratories have suggested that CRD-BP homologues or orthologues function in localizing specific mRNAs to specific regions of the cytoplasm. One CRD-BP homologue initially was characterized in chicken fibroblasts as the b-actin mRNA "zipcode-binding protein" (ZBP).12 This protein binds to a segment of the 3’-UTR of b-actin mRNA that is required to direct the mRNA to the leading edge of the fibroblast (hence the name zipcode binding). Another homologue binds to Vg1 mRNA in the cytoplasm of Xenopus (toad) oocytes and to the transcription factor IIIA gene.16-19 This protein, the Vg1 RNA-binding protein (Vg1 RBP), binds to an element in the 3’-UTR that directs the mRNA to the vegetal pole of the oocyte. Surprisingly, the ZBP and the zipcode interact with microfilaments, while the Vg1 RBP directs Vg1 mRNA to bind to microtubules. The basis for this difference has not been explained.
Still another CRD-BP orthologue was identified based on its capacity to bind to the 5’-UTR of insulin-like growth factor II mRNA.13 In this case, mRNA binding does not stabilize the mRNA but rather represses its translation. It is important to note that there is little sequence similarity among the sequences to which these various proteins bind. Therefore, it will be important to address the basis for mRNA-CRD-BP family interactions in different cell types and organisms.
Summary
Other important questions need to be answered in order to understand the intracellular function(s) of the CRD-BP and its relatives. Is the CRD-BP bound to all c-myc mRNA molecules in the cell? If so, what regulates its affinity for the mRNA, and does it uncouple from the mRNA when cells differentiate or are under stress? One clue to function of the c-myc mRNA-binding family member (CRD-BP) came from the observation that the CRD-BP is developmentally regulated. It is expressed in fetal and neonatal rats but not in adult animals.20 To our knowledge, it is the first mammalian mRNA-binding protein known to be expressed abundantly only in a fetal/neonatal tissue and not in normal adult tissues. It also is known to be expressed in many tissue culture cells lines, which by definition, are transformed. However, its expression is not linked to the replication of at least some normal adult cells, because it is not detected in adult hepatocytes that have been induced to divide at a rapid rate.20
The findings that the CRD-BP is expressed in fetal life and also in transformed tissue culture cells suggested that its expression might be reactivated in adult cancers. To test this hypothesis, tissues from 40 individuals with breast cancer were analyzed for CRD-BP gene copy number using fluorescence in situ hybridization. The gene was moderately amplified in 12 of the 40 cases.11 In two other cases, the gene was highly amplified (14.4 and 20 copies) and appeared to be on double minute chromosomes. Despite their proximity on chromosome 17, the CRD-BP and HER-2/neu genes can be amplified independently. These findings might be significant with respect to the pathogenesis of breast cancer, since amplification of a gene with the capacity to up-regulate c-Myc abundance could accelerate breast cancer. Future studies will need to assess whether the CRD-BP is overexpressed in neoplastic tissues. If so, such a finding might have important diagnostic, prognostic, and eventually, therapeutic implications. (Dr. Ross is Professor of Oncology, McArdle Laboratory for Cancer Research, University of Wisconsin—Madison.)
References
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