KSHV initiates global decay of cellular mRNAs via expression of the virus-encoded ShutOff and Exonuclease (SOX) protein [3
]. Unlike the virion shutoff protein (VHS) of the related herpes simplex virus [4
], SOX itself does not possess any demonstrable nuclease activity [5
], and so how it induces mRNA decay is of considerable interest. In addition, bioinformatic analyses fail to identify any protein-protein interaction domain that would provide a clue to possible co-effectors of SOX-induced mRNA degradation. Thus, Lee and Glaunsinger [2
] had relatively little to guide them as they set out to define the mechanism of SOX-induced RNA decay.
Through a careful analysis of mRNA modifications, localization, and RNA-binding proteins during SOX-induced mRNA degradation, Lee and Glaunsinger made four key observations using a series of transfections and viral infections in human 293T and TIME (telomerase-immortalized microvascular endothelial) cells. First, they documented a clear increase in the size of the poly(A) tail of target RNAs in the presence of SOX that correlated with a decrease in the relative stability of the transcripts. Presumably this is due to the addition of adenosines, although other nucleotides cannot formally be ruled out [6
]. Second, PAPII, the major poly(A) polymerase in the cell that is responsible for the initial mRNA polyadenylation event, was required for this hyperadenylation. This suggests that the PAPII is involved in the hyperadenylation, although it is not entirely clear whether its role is simply to provide the poly(A) tail to be extended or if it is directly responsible for adding the extra 3' nucleotides. Another protein that influences the primary polyadenylation event, the nuclear poly(A)-binding protein PABPN1 [7
], is also required for SOX-mediated mRNA hyperadenylation and decay. Third, there was a dramatic increase in poly(A)+
RNAs in the nucleus, suggesting that the hyperadenylation occurred on many different mRNAs and that an mRNA-trafficking pathway was probably being affected. Fourth, in the presence of SOX, the cytoplasmic poly(A)-binding protein PABPC1 was dramatically relocalized to the nucleus. A similar relocalization of PABPC1 to the nucleus has also been observed in patient-derived KSHV-infected cell lines [8
]. Movement of PABPC1 to the nucleus was directly correlated with the ability of SOX protein to induce decay of cytoplasmic RNAs. Furthermore, knockdowns of PABPC1 by RNA interference (RNAi) reduced the ability of SOX to induce RNA turnover. Finally, reporter mRNAs (made using ribozyme technology) that lacked a 3' poly(A) were immune to SOX-mediated RNA degradation, directly correlating hyperadenylation with SOX-mediated decay. Interestingly, histone mRNAs that naturally lack a poly(A) tail can still be degraded in a SOX-dependent fashion even though they are not hyperadenylated. Thus, whereas the bulk of mRNA decay mediated by SOX involves hyperadenylation and PABPC1 relocalization, alternative degradation pathways appear to exist.
Because hyperadenylation of RNAs has been associated with nuclear surveillance for RNA quality in yeast [9
], and to a lesser extent in mammals [11
], an attractive hypothesis is that SOX is causing the cell's quality control/RNA surveillance machinery to degrade normal mRNAs in some fashion, perhaps by reorganizing the structure of messenger RNA ribonucleoprotein (mRNP) particles. Although this idea is consistent with the PABPC1 relocalization to the nucleus, it should be emphasized that it is currently unclear whether this relocalization is a cause, or a consequence, of SOX-induced RNA degradation. The SOX protein does not possess known interaction domains for poly(A)-binding proteins (for example, PAM2 [13
]), nor do SOX and PABPC1 co-immunoprecipitate. Thus, SOX is likely to modulate PABPC1 localization via an indirect mechanism.