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Sequence and structural requirements for Y RNA cleavage

Periodic Reporting for period 1 - YRNAcleave (Sequence and structural requirements for Y RNA cleavage)

Reporting period: 2016-08-01 to 2018-07-31

"Small non-coding RNAs play important roles in gene expression regulation. Initially the attention was focused on the 21-24-nucleotide small RNAs such as microRNAs but later on deeper sequencing experiments using next generation sequencing revealed a slightly longer class of small RNAs that are 30-34 nucleotides long. These longer small RNAs are often generated from known non-coding RNAs such as tRNA or snoRNA. The biogenesis of these longer small RNAs seems to be diverse and is not well understood. The host laboratory have characterised the biogenesis of such longer small RNAs generated from Y RNAs in mammalian cells and found that it is different from microRNA biogenesis but also from the way tRNA derived small RNAs are produced.

Y RNAs are ~100 nt long molecules, first discovered owing to their association with the autoimmune proteins Ro60 and La. The four Y RNAs (Y1, Y3, Y4 and Y5) show high evolutionary conversation in the animal kingdom, with humans possessing the full complement of Y RNA genes and some species, such as mouse, only retaining the Y1 and Y3 RNA genes. Y RNAs bind a range of proteins within the cell, notably the toroid shaped protein Ro60 involved in RNA quality control, and the La protein which has a range of implicated functions (Figure 1). Not all Y RNAs however are found within ribonucleoprotein complexes. The non-protein bound Y RNAs have been shown to be essential for the initiation of chromosomal DNA replication by acting as licensing factors in post-midblastula transition (MBT) stage cells.

The overall objective of the project is to understand the process of Y RNA fragmentation in human cells. The project consisted of four specific objectives: Objective 1 Validation of sequencing results for pools 1-3, Objective 2 Mutagenesis of 5' arm of Y5 RNA, Objective 3 Validation of mutations in the Y5 5' sRNA region and Objective 4 Testing ""designer"" mutants. We describe the results and conclusions in the next section.

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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.
We described the results in the previous section. As this is a basic research project there isn't an immediate socio-economic impact of the work. Understanding the fragmentation of Y RNAs may have an impact in the future as it is important to understand the molecular details of cell death.
secondary structure of Y5 RNA and bound proteins
Five-nucleotide regions were mutagenised in six pools