| description |
In the fission yeast Schizosaccharomyces pombe, the RNAi machinery is required to generate small interfering RNAs (siRNAs) that mediate heterochromatic gene silencing. Efficient silencing also requires the TRAMP complex, which contains the non-canonical Cid14 polyA polymerase and targets aberrant RNAs for degradation. Here we use high-throughput sequencing to analyze Argonaute-associated small RNAs (sRNAs) in both the presence and absence of Cid14. Most sRNAs in fission yeast start with a 5’-uracil, largely because these are loaded most efficiently into Argonaute. In wild-type cells, most sRNAs match to repeated regions of the genome, whereas in cid14 cells, the sRNA profile changes to include major new classes of sRNAs originating from ribosomal RNAs and a tRNA. Thus, Cid14 prevents certain abundant RNAs from becoming substrates for the RNAi machinery, thereby freeing the RNAi machinery to act on its proper targets.Overall design: Ago1-associated RNA was isolated as in Bühler, et al, (Cell, 2007), and 20-30nt long RNAs were PAGE purified. The eluted small RNAs were cloned based upon the pre-activated, adenylated linkering method using a mutant T4 RNA ligase (Rnl2(1-249)). Single stranded DNA suitable to go directly into the emulsion PCR step of 454 pyrosequencing was generated as described in Margulies, et al (Nature, 2005). |