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Knock out mice that are mutant for a gene product X die. Variants of the X gene that were reintroduced through homologous recombination indicate that variant proteins that lack the C-terminal region of the protein cannot rescue the lethality (did not allow them to live), and always localized to the cytoplasm. Those that retained the C-terminal region rescued the lethality (conferred viability!) and were consistently localized within the nucleus. RNA-seq analysis of the mutant cells vs the wild-type cells indicated that the expression of many genes that are essential for neural function was reduced in the knock-out mutant cells.
The N-terminal region of Protein X is 100% conserved between mouse and humans at both the amino acid and the
5’-AUGUUUACAGAGGGGAAU... -3’
Q3a) Using a total RNA sample obtained from human cells, what is the first primer you would use in an RT reaction to generate a single stranded cDNA that corresponds to this mRNA? (Write a 10 nt sequence of the primer from 5'-3')
Q3b) Design a 12 nt upstream primer that would allow you to amplify a full length double stranded cDNA that corresponds to Human Protein X by PCR after having performed the initial RT reaction (Write the 12 nt sequence of the primer from 5'-3')
Q3c) In what cellular compartment is the mouse Protein X required in order to fulfill its essential function?
Q3d) What motif could be present to direct this protein to its correct destination?
Q3e) From the cellular localization data and the RNA-seq analysis, what is the likely function of Protein X in regulating the expression of these neuronal genes?
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