The impact of codon usage from bicistronic mRNA on their transcriptomic, post-transcriptomic and proteomic regulation in human cells

ABSTRACT: A large amount of virus infecting humans expresses polycistronic mRNAs on which viral ORF display codon usages (CU) dissimilar to the average human ORF CU. Among those viruses, oncogenic Human Papillomavirus (HPV) are responsible for 5% of all cancer cases. It is known that CU is a major step of both quantitative and qualitative translation regulation. On top of that, human translation machinery mainly come across monocistronic mRNAs. The translation of downstream ORF on polycistronic mRNAs is generally ill-defined, relying on non-canonical translation mechanisms. CU variation could also modulate downstream ORF translation. We believe that poorly matching CU between polycistronic viral mRNAs and the translation machinery of its host may have conferred oncogenic HPVs an evolutionary advantage by reducing viral protein expression, facilitating immune escape, persistence of infection and establishment of carcinogenesis. To unravel variables influencing viral mRNAs regulation, we expressed in the U-2 OS cell line 13 versions of an shble_gfp bicistronic mRNA which i) mimic the e6_e7 mRNA from HPVs, ii) display 13 different CU to code the same SHBLE protein and, iii) for 5 version undergo splicing in shble ORF (which was latter castrated given us a total of 18 constructs). We then quantified, mRNA expression, splicing efficiency, GFP fluorescence and, SHBLE and GFP expression via mass-spec. We first confirmed that monocistronic gfp mRNAs are a 100 times more efficiently translated than any shble_gfp bicistronic mRNAs. No impact was found of shble CU on mRNA transcription levels. The 5 versions comparison between spliced vs. mutated revealed a positive impact of shble splicing on GFP translation. Next, we showed that CU greatly affect the expression of SHBLE. Finally, we strikingly showed that the variation in CU and SHBLE expression does not significantly affect downstream GFP expression, suggesting that GFP is translated via leaky scanning.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Bone Marrow

DISEASE(S): Osteosarcoma

SUBMITTER: Functional Proteomics Platform FPP  

LAB HEAD: Ignacio G. BRAVO

PROVIDER: PXD047576 | Pride | 2025-05-06

REPOSITORIES: Pride