Tumor suppressor p53 is one of the most extensively investigated molecules. It has been implicated in various processes but its role in cell cycle arrest and apoptosis is well established (reviewed in 1
). p53 levels are predominantly regulated via its protein stability that involves modulation via ubiquitination and proteasomal degradation. In this regard, several ubiquitin ligases have been implicated to promote p53 ubiquitination and subsequent degradation (3
). Pirh2 is one of the key ligases that promote p53 ubiquitination and degradation (4
). Pirh2 is a RING domain ligase and a direct transcriptional target of p53 and thus, believed to function via a mechanism involving negative feedback loop to inhibit p53 function (4
We have recently identified and characterized two novel isoforms of Pirh2 named Pirh2B and Pirh2C (8
). Accordingly, we reclassified full-length Pirh2 as Pirh2A. Both Pirh2B and Pirh2C harbor truncated versions of the RING domain and lack the ubiquitin ligase function (8
). Pirh2B lacks 9 amino acids within the RING domain of Pirh2 ligase, whereas Pirh2C is missing the last seven amino acids in the RING domain and the entire downstream carboxyl-terminal sequence (8
). Upon recent database searches, we have noted that in addition to these novel isoforms, there exists yet an additional isoform of Pirh2. For example, expressed sequence tag (EST) database revealed the existence of ESTs that could correspond to an additional Pirh2 isoform.
To experimentally verify that indeed these ESTs correspond to a different isoform of Pirh2, we obtained (OriGene, Rockville, MD) cDNA clone of one of the ESTs (GenBank accession number DR000555) and sequenced it in its entirely. We compared its nucleotide sequence with the sequences of Pirh2 isoforms A, B and C and noted that it harbored an additional nucleotide “A” that was predicted to shift the reading frame and generate a premature stop codon ( and ). Thus, translation of the cDNA sequence predicted a truncated Pirh2 protein of 75 amino acids () with molecular mass of 8493.74 daltons. We further noted that the predicted truncated protein harbored 67 N-terminal amino acids as identical to those in Pirh2A, Pirh2B and Pirh2C and had 8 additional unique amino acids (N S T C P T D L) followed by a stop codon ().
Nucleotide sequence of EST clone corresponding to Pirh2D
The predicted amino acid sequence of Pirh2D
To verify that this EST would encode the truncated protein of expected size, we transiently transfected human embryonic kidney cells 293 with the expression vector carrying this cDNA; vector-only was used as a control. Cells were harvested approximately 48 hours post-transfection and Western blot analysis was performed using anti-Pirh2 antibody. Results in indicate that the EST clone (GenBank accession number DR000555) encodes a truncated protein of expected size and thus, an additional isoform of Pirh2 that we have named Pirh2D. What is the mechanism that would lead to the insertion of an additional nucleotide in the sequence of Pirh2D remains unclear at the present time. The fact that three ESTs corresponding to Pirh2D with identical insertion of “A” are present in three independent libraries generated from three different tissues including testis (GenBank accession number DB455131), fetal brain (GenBank accession number DR000555) and hypothalamus (GenBank accession number DB488919) would indicate that it is not result of cloning or sequencing artifact.
Western blot analysis showing expression of exogenous Pirh2D in 293 human embryonic kidney cells
The Pirh2 gene is composed of 9 exons and we have recently reported that Pirh2B lacks complete exon 7 most likely due to alternative splicing (8
). The exon 7 is composed of 27 nucleotides encoding 9 amino acids as part of the RING domain (8
). Prih2C, by contrast, appears to arise due to usage of alternative donor site during splicing, which results in the introduction of 28 base-pairs of intronic sequence between coding exons 7 and 8 and generation of a premature stop codon (8
). In the case of Pirh2D, there is an insertion of a nucleotide “A” that shifts the reading frame resulting in 8 unique amino acids and a premature stop codon generating a truncated protein of 75 amino acids.
What might be the function of Pirh2D is a question that awaits further investigation. We have recently reported that Pirh2B and Pirh2C are not capable of auto-ubiquitination and thus, lack the ubiquitin ligase function (8
). However, we noted that expression of exogenous Pirh2B and C decreased the levels of endogenous wild type p53 in human colon cancer cells and also inhibited p53 transcriptional activity (8
). Our results further indicated that both Pirh2B and C also promoted intracellular ubiquitination of p53 and exhibited interactions with MDM2. Pirh2D, the smallest of all isoforms, also lacks the C-terminal RING domain and is predicted to lack ligase function (8
). However, whether it is capable of functioning in a manner similar to Pirh2B and Pirh2C is an interesting question that remains to be investigated. Future studies will provide further insight into the expression profile and relative abundance of Pirh2D in various tissues and also its function.