RNA polyuridylation: regulation and consequences

While the DNA in our cells contains the information that controls their behaviour, this information has to be copied into RNA 'messages' before it can be decoded, for example to make proteins. The information content of each message, copied from the DNA template, is encoded in a long string of chemical letters, of which there are four types: A, U, C and G. For some time it has been known that the end of most RNA messages consists of a string of 'A' letters that are not encoded in the DNA, but added after the message has been copied. The length of this A-tail determines how long the message lasts before being destroyed, and how efficiently it is decoded to make protein. We have recently discovered that messages can instead have tails made mostly of 'U' letters, and that this is something that happens in organisms as distantly related as yeasts and humans. This project aims to investigate the function of these U-tails, and to determine how their synthesis is regulated. Our preliminary results suggest that, in the longer term, drugs that interfere with the addition of U-tails to RNAs might inhibit the growth of cancer cells.

Nuclear poly(A) polymerase (PAP) polyadenylates nascent mRNAs to promote their nuclear export, stability and translation, while the related cytoplasmic polymerase GLD-2 activates translation of de-adenylated mRNAs. We have recently characterized the biochemical activity of fission yeast Cid1, a cytoplasmic PAP/GLD-2-related enzyme implicated in cell cycle checkpoint controls. Surprisingly, while acting as a PAP in vitro, Cid1 has far more robust poly(U) polymerase (PUP) activity, especially when isolated in native multi-protein complexes. This PUP activity is conserved in human cells, where the Cid1-related protein Hs2 [ZCCHC6] shows similar nucleotide preferences. Our preliminary data indicate that mRNA polyuridylation occurs in vivo. The biochemical basis for the increased PUP versus PAP activity of purified Cid1 multi-protein complexes will be investigated by evaluating the contributions made by Cid1-interacting proteins, possible post-translational modifications and associated nuclease activities. Microarray hybridisation using oligo(dA)-primed cDNA will be used to identify polyuridylated RNAs in fission yeast and human cells, and the consequences of mRNA polyuridylation will be investigated using MS2 tethering and in vitro RNA degradation assays. Together, these studies will allow us to investigate the hypothesis that U-rich Cid1/Hs2-generated tails form the basis of a novel, conserved level of RNA regulation.