The HPV-16 E7 oncoprotein is expressed mainly from the unspliced E6/E7 transcript in cervical carcinoma C33-A cells

Abstract

The HPV-16 E6/E7 early transcripts are first produced as bicistronic or polycistronic mRNAs, and about 90% of the original pre-mRNA is spliced to produce three new alternative mRNAs. HPV-16 spliced transcripts are expressed heterogeneously in tumors and cell lines. Our results suggest that suboptimal splicing acceptor sites in E6/E7 intron 1 and the differential expression of splicing factors are involved in the production of the heterogeneous splicing profile in cell lines. The unspliced pre-mRNA and the alternative spliced transcripts contribute differentially to the production of E7 in stably transfected C33-A cells. The highest level of E7 was produced from the least prevalent transcript, the unspliced E6/E7(pre-mRNA). The order of relative expression of E7 was unspliced E6/E7(pre-mRNA) > E6*I/E7 > E6*II/E7. Our findings suggest that E6/E7 alternative splicing may be a mechanism for differential expression of the E6 and E7 oncoproteins, which also affects the expression of their targets, the proteins p53 and pRb.

Differential In Vitro Immortalization Capacity of Eleven, Probable High-Risk Human Papillomavirus Types

“…Whether splicing of E6 enhances the translation efficiency of E7 is still controversial. Whereas in some studies an increase in the efficiency of E7 translation was reported (25)(26)(27), in other studies, E7 is translated equally efficiently from spliced and unspliced transcripts (28,29). Nevertheless, E6* has been found to function independently of E6 in the transformation process (30,31).…”

Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16

“…References are represented by superscript lowercase letters, where a refers to the detected mixed A and B spliceosomal complexes in Bennett et al [60]; b is that detected in B complexes in Deckert et a l. [34]; c, detected in A, B, or C complexes in Herold et al [59]; d, detected in A, B, or C complexes in Agafonov et al [35]; e, detected in C complexes in Jurica et al [17]; f, detected in C complexes in Yang et al [54]; g, detected in mixed complexes in Zhou et al [38]; h, Rasche et al [61]; i, detected supra-spliceosomal (SS) complexes isolated in bulk in Chen et al [33]; and j, detected in Behzadnia et al [52]; spliceosomal complex = sp1 complex. transcripts are heterogeneously expressed in tumor cells, but the cause of the heterogeneity is poorly understood [21,22]. Recognition of the In-1 splicing sequences is probably affected by the availability of a set of splicing factors, which are largely unknown.…”

“…The HPV-16 E6/E7 pre-mRNA is alternatively spliced and produces 4 transcripts. One of them is the unspliced pre-mRNA self, which is the most important of these mRNAs, since it is the only source of the full length E6 oncoprotein [21,25]. In-1 is constituted by one donor site and three acceptor sites, but only the first two acceptors are located in the E6 gene.…”

“…For C7m the HPV-16R/HPV16M5H primer pair was used and it produces a 147 nt DNA fragment. Constructs C4m and C5m were amplified from the previously reported mutant plasmid pE6/E7SD M [21], using the following primer pairs: SA-A Ext Fw/HPV-16 3AMR and FE6MUT226 Eco/HPV-16 3AMR, respectively. Construct C6m was produced by joining the DNA fragment of construct C2m, with an amplicon that contains the mutated acceptor C; previously, a Not I restriction site was introduced into the B-acceptor site by using the primer pair HPV-16R/HPV-16A3SSMR.…”

“…The heterogeneous profile seems to be promoted by two conditions: intrinsic variation in the level of some splicing factors in the tumor cells [21] or a differential binding of splicing factors to polymorphic variants of the HPV-16 [22]. Unfortunately, both the mechanisms and splicing factor that facilitate this heterogeneous expression are largely unknown.…”