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. 2022 Mar 2;50(5):2782–2806. doi: 10.1093/nar/gkac132

Figure 6.

Figure 6.

hnRNP D40 interacted with HPV16 E6E7 RNA sequences in vitro and influenced HPV16 E6E7 mRNA processing. (A) RNA oligonucleotide-mediated pull-down assay using sequential short RNA oligos spanning the entire HPV16 E6- and E7- coding regions. The numbers in each RNA-oligo name represent HPV16 nucleotide positions and refer to nucleotide numbers in the reference HPV16 genome (GenBank: K02718.1). Thick double-headed arrow: intronic region of E6/E7 mRNAs located between SD226 and SA409. (B) Schematic representation of RNA splicing in the HPV16 E6 and E7 coding region. Intron-retained E6 mRNA is depicted as well as spliced HPV16 mRNAs 226∧409, 226∧526 and 226∧742. Box with S: previously identified E6/E7 mRNA splicing silencer element (46). RNA oligo sequences are available in Supplementary Table S3. (C) The effect of hnRNP D40 on the splicing of HPV16 E6E7 mRNA produced from pC97ELsLuc as well as shorter plasmids pX656 and pX478 was monitored by RT-PCR. The schematic maps of pX656 and pX478 plasmids and the positions of RT-PCR primers are shown in Supplementary Figure S5. (D) The percentage of intron-retained E6 mRNA over a total sum of all spliced isoform quantitated from (C) are shown. (E) HPV16 E6 and E7 proteins expressed in HeLa transfected with pC97ELsLuc together with indicated wild-type or mutant hnRNP D40. Western blotting was performed using anti-HPV16 E6 or E7 specific antibodies. (F) HPV16 E6 and E7 proteins produced in 293T cells transfected with pXH856F, which encodes intact HPV16 E6- and E7- coding regions, or with pXH856SDmF, which contains a mutationally inactivated SD226 splice site, in the absence (−) or presence (+) of cotransfected hnRNP D40 expression plasmid. Western blotting was performed with anti-HPV16 E6 or E7 specific antibodies. Wild-type SD226 (GAG|GUAUAUGA: a vertical line indicates 5′ splice site) was changed to (GAG|GCCUAUGA: underline indicates changed nucleotide) in SD226 mutant. Schematic maps of pXH856F and pXH856SDmF are shown in Supplementary Figure S5. (G) Subcellular distribution of intron-retained E6 mRNAs produced from pXH856SDmF in the absence (−) or presence (+) of hnRNP D40 plasmid. Nuclear and cytoplasmic fractions were prepared from the transfected cells and RNA was extracted and subjected to HPV16 RT-PCR using primers TotalE6F and 757AS (for primer location, see Figure 1C and Supplementary Figure S5E). Cell fractionation was validated by RT-PCR analysis of unspliced actin RNA located exclusively in the nuclear fraction while spliced actin mRNAs found in both fractions. (H) qPCR using cDNA samples from (G) to evaluate levels of intron-retained E6 mRNAs in nuclear and cytoplasmic fractions in the absence or presence of hnRNP D40. Primers TotalE6F and 234AS were used (for primer location, see Figure 1C and Supplementary Figure S5E). (I) Nuclear (N) and cytoplasmic (C) distribution of HPV16 alternatively spliced mRNAs produced from pXH856F in the absence (−) or presence (+) of hnRNP D40 plasmid. RT-PCR was performed with primers: 97S+757AS (top panel) or 97S+438AS (second panel). Primer locations are shown in Figure 1C and Supplementary Figure S5D. (J–L) Nuclear (N) and cytoplasmic (C) distribution of HPV16 alternatively spliced mRNAs produced from pC97ELsLuc in the absence (−) or presence (+) of cotransfected hnRNP D40 plasmid. RT-PCR was performed with the following primers: (J) 97S+880AS, (K) 97S+438AS or (L) 773S+E2QAS. (M) Nuclear (N) or cytoplasmic (C) distribution of HPV16 alternatively spliced mRNAs produced from HPV16 plasmid pX478 in the absence (−) or presence (+) of cotransfected hnRNP D40 plasmid, followed by RT-PCR using primers, 97S+438AS. Schematic map of plasmid pX478 and location of HPV16 RT-PCR primers are shown in Supplementary Figure S5C. (N–P) Percentage of cytoplasmic intron-retained E6 mRNAs over the total sum of nuclear and cytoplasmic intron-retained E6 mRNAs in the absence (−) or presence of hnRNP D40 quantitated on (N) pXH856F from (I), (O) pC97ELsLuc from (K) or (P) pX478 from (M). Student t-test was executed and obtained P values were displayed. n.s., no significance.