Comparative genomic analysis of the 3' UTR of human MDM2 identifies multiple transposable elements, an RLP24 pseudogene and a cluster of novel repeat sequences that arose during primate evolution.

The MDM2 oncogene is a negative regulator of the p53 tumour suppressor. This relationship appears to have originated over a billion years ago. The human MDM2 gene encodes a variety of mRNAs with exceptionally long 3'UTRs (up to 5.7 kb); however, it was unclear whether MDM2 3'UTRs from other species are similarly long or conserved at the sequence level. Here, we report that all but one of the primate species most closely related to humans (greater and lesser apes) have similarly long 3'UTRs with high sequence similarity across their entire length. More distantly related species (Old world monkeys and new world monkeys) tend to have shorter MDM2 3'UTRs homologous to the corresponding position of the human MDM2 3'UTR while non-primate species exhibit little similarity at all. Remarkably, DNA sequences downstream of the shorter primate 3'UTRs are syntenic with distal regions in the human and other ape MDM2 3'UTRs. These homologous non-transcribed intergenic and transcribed 3'UTR-encoding regions are comprised of a variety of transposable elements, an RLP24 pseudogene and a cluster of novel repeat sequences suggestive of another unknown transposable element. Our analysis suggests that the primary difference between long and short MDM2 3'UTRs is a switch in polyA site usage to include conserved transposable elements that remain intergenic in more distantly related primates. It will be important to determine the relative contribution of these elements to post-transcriptional and translational regulation of MDM2 and hence p53-mediated tumour suppression.

A Temperature Sensitive Variant of p53 Drives p53-Dependent MicroRNA Expression without Evidence of Widespread Post-Transcriptional Gene Silencing.

The p53 tumour suppressor is a transcription factor that can regulate the expression of numerous genes including many encoding proteins and microRNAs (miRNAs). The predominant outcomes of a typical p53 response are the initiation of apoptotic cascades and the activation of cell cycle checkpoints. HT29-tsp53 cells express a temperature sensitive variant of p53 and in the absence of exogenous DNA damage, these cells preferentially undergo G1 phase cell cycle arrest at the permissive temperature that correlates with increased expression of the cyclin-dependent kinase inhibitor p21WAF1. Recent evidence also suggests that a variety of miRNAs can induce G1 arrest by inhibiting the expression of proteins like CDK4 and CDK6. Here we used oligonucleotide microarrays to identify p53-regulated miRNAs that are induced in these cells undergoing G1 arrest. At the permissive temperature, the expression of several miRNAs was increased through a combination of either transcriptional or post-transcriptional regulation. In particular, miR-34a-5p, miR-143-3p and miR-145-5p were strongly induced and they reached levels comparable to that of reference miRNAs (miR-191 and miR-103). Importantly, miR-34a-5p and miR-145-5p are known to silence the Cdk4 and/or Cdk6 G1 cyclin-dependent kinases (cdks). Surprisingly, there was no p53-dependent decrease in the expression of either of these G1 cdks. To search for other potential targets of p53-regulated miRNAs, p53-downregulated mRNAs were identified through parallel microarray analysis of mRNA expression. Once again, there was no clear effect of p53 on the repression of mRNAs under these conditions despite a remarkable increase in p53-induced mRNA expression. Therefore, despite a strong p53 transcriptional response, there was no clear evidence that p53-responsive miRNA contributed to gene silencing. Taken together, the changes in cell cycle distribution in this cell line at the permissive temperature is likely attributable to transcriptional upregulation of the CDKN1A mRNA and p21WAF1 protein and not to the down regulation of CDK4 or CDK6 by p53-regulated miRNAs.