Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

Abstract

HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5′ splice sites (5′ ss) and three 3′ splice sites (3′ ss) normally used in HPV16+ cervical cancer and its derived cell lines. The choice of two novel alternative 5′ ss (nt 221 5′ ss and nt 191 5′ ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5′ ss and nt 409 3′ ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3′ ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3′ ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of 91QYNK94 to 91PSFW94 displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

“…Looking forward, the identification and characterization of the HPV18 ESE and ESS in this report may in due course offer potential therapeutic targets to overcome HPV-related cancer. 3= end was attached to a U1 binding motif (11 nt) (gray boxes) to enhance its splicing efficiency (20,32). The numbers are the nucleotide positions in the virus genome.…”

“…Alternative RNA splicing of the HPV polycistronic pre-mRNAs plays a crucial role in regulation of viral gene expression (2,3). Although the molecular mechanisms that regulate alternative RNA splicing of bovine papillomavirus type 1 (BPV-1) (4-11) and HPV16 pre-mRNA transcripts (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) have been extensively studied in the past, a full transcription map of HPV18 in productive infection, the second most prevalent high-risk HPV genotype in association with cervical cancer (23), was constructed only recently (24), and the mechanistic regulation of HPV18 RNA splicing remains poorly investigated. HPV18 pre-mRNAs are transcribed mainly from a major early promoter, P 55/102 , or a major late promoter, P 811 , although a few other, weak promoters exist in the virus genome (24,25).…”

Human Papillomavirus Type 58 Genome Variations and RNA Expression in Cervical Lesions

“…HPV16 plasmid DNA (0.2 ng) served as a positive PCR control with the primer pair Pr122 and Pr821 (Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a DNA loading control with the primer pair oZMZ269 and oZMZ270 (24). PCR products were digested overnight by SwaI at 25°C, by BstEII at 60°C, or by MfeI at 37°C.…”

Alternative RNA splicing of KSHV ORF57 produces two different RNA isoforms

“…The first intron in the 5′ half of ORF57 RNA is a constitutive intron, with a small (114 nts), capped exon immediately upstream and thereby, its splicing can be efficiently promoted by the capping machinery cross over the exon (Konarska, Padgett et al, 1984;Lewis, Izaurralde et al, 1996;Zheng, Tao et al, 2004). In addition to the suboptimal feature of the intron 2, a large size (749 nts) of the internal exon 2 upstream of the intron 2 may also prevent recognition of the intron 2 5′ splice site by the cellular splicing machinery It has been well documented that the size of an internal exon affects cross-talk (exon definition) of a 3′ splice site and a 5′ splice site over the exon and this cross-talk is limited by an oversized exon (>500 nts) (Robberson, Cote et al, 1990;Sterner, Carlo et al, 1996;Zheng, 2004;Ajiro, Jia et al, 2012). Moreover, the intron 2 3′ splice site AG in ORF57 is preceded by unfavorable A and may also contribute to a low level of intron 2 splicing during native KSHV infection (Fig.…”