Question

How could an early stop mutation lead to an N-terminal truncated protein? What experiments would you conduct to confirm this hypothesis? (This is in regard to an article on BRCA 1 mutations, but this question is in general)

Answer 1

N-terminal truncation occurs due to the changes in the structural gene by a premature stop codon (by a frameshift or non-sense mutation) that terminates the elongation of the specific gene. In the case of early stop mutation or non-sense mutation may interfere with the translation, and might terminate it. But the reinitiation ribosomes can occur downstream of the start codon, AUG. If the stop codon was proximal to the start codon, the reinitiation efficiency is normally high, and this leads to the expression of the N-truncated protein. Though reinitiation occurs, the rate of reinitiation is lower than the original rate of initiation. Leaky scanning is associated with the expression of the N-truncated protein post the stop codon.

Western blotting, real-time quantitative PCR (RT- qPCR), and RNA interference studies can help in analyzing the expression of the N-terminal truncated protein.

(i) Western blot studies using the antibody binding can show the variants of the N-truncated protein. the N-truncation is dependent on the loci on the non-sense mutation to the start codon. Following the stop codon, the ribosomes can scan an AUG and can reinitiate an N-truncated protein, which can be observed by the protein variants in the Western blotting.

(ii) The RNA that has been isolated can be converted to cDNA using the reverse transcription, and the RT- qPCR can be performed for analyzing the variants. The ratio between the normal expression and truncated expression can be analyzed to know about the promoter variation, level of truncated translation, etc.

(iii) Cell line studies for N-truncated proteins can be transfected with specific siRNA directed against the mRNAs that get transcribed.

Ref: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614817/