To make the production of transcripts A, B, and C sensitive to eIF4E-dependent mRNA export, the first thing I will add is 1) A coding sequence of approximately 50 discrete nucleotide elements at their 3'UTR known as the eIF4-4E sensitivity element (4E-SE).• The presence of 4E-SE acts as a USER code for eIF4E-sensitive transcripts and therefore increases mRNA export from the nucleus to the cytoplasm.• eIF4E-SE containing mRNA depends on CRM1. • To test for increased mRNA export, we can use an inhibitor such as LeptomycinB (LMB), with an appropriate amount of vehicle control (ethanol), to see if 4E-SE dependent nuclear export of mRNA and eIF4E increased compared to inhibitor-treated cells by separately collecting nuclear and cytoplasmic fractions from these cells. More detailed analysis can be performed by qRT-PCR or Northern blot. (Culjkovic B, et al Cell Cycle. 2007, Journal of Oncology. 2009, Cell Reports. 2012).2) Second, I would add a complex coding sequence to the 5'UTR region, thus making it an export-sensitive mRNA and helping it easy translation.• Sensitive transcripts of eIF4E will have a higher ratio of ribosome/mRNA level, which helps in efficient translation.• In the case of eIF4E, the 5_UTR complex and 4E-SE in the 3_UTR can be considered USER codes for a efficient translation and export.• To verify the increase in translation efficiency, we can perform the analysis of the polysome load which will be associated with heavier polysomes.b) (30 points) If we wanted to suppress the production of A at the translational level , while increasing mRNA B and C levels (post-transcriptional), what could you do? (You are not allowed to use siRNA or miRNA strategies here; but you can design the constructs as you wish. You can also add other... middle of paper... multiple polypeptides simultaneously from a single mRNA (Multiple ribosomal translations of a single mRNA). This can be verified by polysomal loading experiments. Polysomal loading experiments begin with the isolation of mRNAs of interest from cells by immunoprecipitation with mRNA binding or by sucrose density gradient centrifugation based on loading of ribosomes onto mRNAs Untranslated and actively translated mRNAs can be separated by isolating the monosomal and polysomal fractions by centrifugation through sucrose. If the mRNA of our interest has multiple ribosomes bound to it in the polysomal fraction, this can be further used for qRT-PCR analysis and. microarray, extracting RNA or proceeding with hybridization analysis of gradient fractions. The findings could tell us why there is increased production of protein Z without changing RNA levels.