Hello everyone,
I'm doing polysome profiling to answer a specific question.
I study a gene which produce an alternative transcript and it's translation is controversial (extremely lucky if you can see the truncated protein by western blotting after a harch MG132 treatment).
I know the unspliced transcrit is well translated and I want to know if the spliced one is translated as well.
So I transfected cells to express the two transcripts and I realised a polysome profiling experiment. And the transcripts are exactly in the same fractions at the same percentage.
To confirm this result, I did the same experiment but with transcripts with no ATG and I also find the same results... (noATG transcripts are in the same fractions as normal transcripts).
I was expecting the noATG transcirpts to be in the free RNA fraction but they are in the monosome ans polysome as well.
Anyone has an explanation ?
Thanks for the help you can provide !
You may have to consider a functional non-canonical ORF in your transcript. Just an idea. See e.g.: Article Non-AUG translation: A new start for protein synthesis in eukaryotes
Thanks for your answer Uwe Borgmeyer !
My transcript also has 13 AUG in total and I only mutated two of them.
Even if my deltaAUG transcript is not translated, maybe ribosomes recognize the other AUG at a given time. And the CHX treatment before polysome profiling could make them stay on my deltaAUG transcript.
Anyway those deltaAUG transcript don't seem to be good negative controle for this experiment...
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