Question

A sequence of a eukaryotic gene (coding strand) is shown below, RNA polymerase recognizes the sequence ‘TATAAT’ and initiates transcription six nucleotides downstream of the sequence. The intron splice sites are CUU (5’ splice site) and AAG (3’ splice site), poly-A tails are added following the sequence AGUUGG. The poly-A tails are 20 nucleotides.

b. If this is an oncogene that is elevated in cancer cells, design two siRNAs to knock down the mRNA, list the sequences of the siRNAs and highlight the targeted regions on the mRNA.

1 GATGAGTTAT AATATTTCTC TCCAGGCATG GAGTATTCCG GTGTGCGATC GCCGTTATCG ATCGATCGAT

71 ATCGATCGAA TCCCCCTTTG GACCACCCTG GGTTGCCCTC TAAGCATAAT TCGTCGTCGT ACAACGCCAT

141 GTTTCTGT AATTAAAATT TGTTTGCCTC AGTTGGATGT

Answer 1

mRNA sequence is identical to the coding strand in 5' to 3' direction and complementary to the non-coding also called the template strand.

The sequence of the unspliced pre-mRNA is given below. The transription initiates 6 nucleotides downstream of the TATAAT sequence in the promoter region. The poly A signal is added after  AGUUGG sequence. All Ts in the given will be replaced by Us in the mRNA

5'CUCCAGGCAUG GAGUAUUCCG GUGUGCGAUC GCCGUUAUCG AUCGAUCGAU AUCGAUCGAA UCCCCCUUUG GACCACCCUG GGUUGCCCUC UAAGCAUAAU UCGUCGUCGU ACAACGCCAU GUUUCUGU AAUUAAAAUU UGUUUGCCUC AGUUGGAAAAAAAAAAAAAAAAAAAA3'

Now from the above sequence of pre-mRNA remove the stretch of sequence from the underlined CCU to underlined AAG. This makes the splice mRNA

5' CUCCAGGCAUG GAGUAUUCCG GUGUGCGAUC GCCGUUAUCG AUCGAUCGAU AUCGAUCGAA UCCCCCAUAAU UCGUCGUCGU ACAACGCCAU GUUUCUGU AAUUAAAAUU UGUUUGCCUC AGUUGGAAAAAAAAAAAAAAAAAAAA 3'

Scan the sequence for AA dinucleotides and the next 19 nucleotides. The GC content should be less than 30- 50%

AA UCCCCCAUAAU UCGUCGUC ( GC content is 52%)

AAUUCGUCGUCGU ACAACGCC ( GC content is 57%)

AAUUAAAAUU UGUUUGCCUC A (GC content is 26%)

We will choose th sequence AA UCCCCCAUAAU UCGUCGUC as the target

Sequnece of siRNA

5' UCCCCCAUAAU UCGUCGUC UU 3' (sense strand)

3' UUAGGGGGUAUUAAGCAGCAG5' (antisense strand)