The Dalmay group previously carried out a highthroughput mutagenesis at the 3' half of Y5 RNA (Figure 2) and initial analysis suggested that the secondary structure of the Y RNA, rather than its sequence, determines where the cleavage happens that liberates the small RNAs from the 3' end of Y RNAs. The first objective of this project was to experimentally validate this. We generated many mutant forms of the Y5 RNA, based on the sequencing result of the mutagenesis experiment, and studied how small RNAs were generated from these specific mutants. The results confirmed that regardless of the sequence, the specific secondary structure was responsible for the cleavage site (objective 1). More specifically, the cleavage happens two nucleotides away from the terminal stem into the internal loop. if we change the sequence such that the stem is a bit longer than the small RNA gets longer as the cleavage still happens two nucleotides away from the stem (and because the stem is longer, the small is RNA longer). Next we carried out a highthroughput mutagenesis at the 5' half of Y5 RNA (Figure 2), transfected the pool of mutants into cells, induced small RNA production by apoptosis and sequenced the generated small RNAs (objective 2). Similarly to the 3' end, some mutants produced many small RNAs and others produced very little. These were confirmed experimentally by testing specific mutants (objective 3). Finally we generated various mutants of Y5 RNA where the mutations were either outside of the mutated regions or on both sides of the stem (objective 4). We concluded from the experiments that the secondary structure plays an important role at the 3' end but not at the 5' half, where the UAUUG motif (the left side of the internal loop, Figure 2) is more important than the secondary structure.
We are writing a manuscript at the moment and we are planning to submit it in about 3 months. Trung presented a poster about the work at the Non-coding RNAs conference in Heidelberg in the summer of 2017.
