2013 Rustbelt RNA Meeting
With the exception of histone mRNAs, 3’end formation of all mRNAs in higher eukaryotes is thought to occur via cleavage and polyadenylation. In studies of miRNA action on target mRNAs in S2 cells, a number of mRNAs were analyzed by using a polyA tail length measurement method. While several mRNAs displayed the expected heterogeneous tail, two mRNAs, those encoding Hid and Reaper appeared to have short (less than 10 nucleotides) polyA tails. This phenomenon was observed when the 3’UTR of Hid and Reaper were appended to luciferase reporter constructs. To distinguish between lack of polyadenylation and deadenylation, we knocked down the deadenylase components CCR4 and POP2. Remarkably, in cells depleted of CCR4 and POP2, both Hid and Reaper mRNAs were found to have normal polyA tails. This result clearly indicated that the major form of Hid and Reaper mRNAs at steady state in S2 cells is deadenylated. Further analysis was done using the Reaper 3’UTR. Systematic deletion and substitution analyses demonstrated that several elements, including two cis-acting sequences (20-24 nucleotides) located 178 bases and 118 bases upstream of an AAUAAA hexanucleotide, the hexanucleotide itself, and bases 73-115 downstream of the hexanucleotide, were necessary and sufficient to cause the deadenylated phenotype. The position of the upstream elements relative to the hexanucleotide is also important. Interestingly, we have also found that the deadenylated Reaper mRNAs are engaged with actively translating ribosomes. Importantly, the deadenylated Reaper mRNAs can resume active translation after exposure to conditions that inhibit initiation of protein synthesis, such as hypertonic stress. We are currently attempting to identify the trans-acting factors interacting with the elements in the Reaper 3’UTR, which are required for maintenance of translating deadenylated mRNAs.
Keywords: deadenylation, translation