Posttranscriptional regulation of mRNA turnover is a major mechanism regulating gene expression (reviewed in Garneau et al., 2007
), and work done in the past several years has elucidated the fundamental mechanisms of decay and has begun to clarify regulation of the decay process and its relationship to translation. The decay of most mRNAs begins with poly(A) shortening, followed by exonuclease-mediated decay involving removal of the cap, 5′-3′ decay catalyzed by Xrn1, and 3′-5′ decay catalyzed by the exosome. The identification in 2003 (Sheth and Parker, 2003
) of processing bodies (P bodies) as focal concentrations of the decapping proteins Dcp1 and Dcp2, Xrn1, and other proteins involved in 5′-3′ mRNA decay led to the early conclusion that these are sites of mRNA decay. More recent work shows P bodies play a larger role in mRNA storage and silencing, and using quite different approaches we (Murray and Schoenberg, 2007
) and others (Stoecklin et al., 2005
) showed that unstable mRNAs can decay simultaneously from both ends and that 5′-3′ and 3′-5′ decay are functionally linked. A notable feature of the exonuclease-mediated decay process is that it acts on mRNAs that are no longer bound by translating ribosomes.
For reasons yet to be determined, some mRNAs decay by endonucleolytic cleavage while they are still engaged by ribosomes and undergoing active translation. Because endonucleolytic decay acts on specific mRNAs their targeting to this pathway must be dictated by sequence elements within the mRNAs that are bound by one or more proteins that recruit the endonuclease to the translating messenger ribonucleoprotein (mRNP). Polysomal ribonuclease 1 (PMR1) was the first mRNA endonuclease to be identified (Dompenciel et al., 1995
), but others include G3BP (Gallouzi et al., 1998
), IRE1 (Hollien and Weissman, 2006
), and a yet to be identified erythroid-specific endonuclease (Wang and Kiledjian, 2000
). PMR1 was initially identified as a polysome-associated ribonuclease activity whose appearance on Xenopus
liver polysomes coincides with the disappearance of serum protein mRNAs during estrogen induction of yolk protein gene transcription (Pastori et al., 1991a
). Subsequent work showed that mammalian PMR1 catalyzes the initial steps in the decay of nonsense-containing β-globin mRNA in erythroid cells (Stevens et al., 2002
; Bremer et al., 2003
). PMR1 is a member of the peroxidase gene family that is made as an 80-kDa precursor (PMR80) that is processed to the active 60-kDa form (PMR60) (Chernokalskaya et al., 1998
). Estrogen has no effect on the amount of this protein in Xenopus
hepatocytes; rather, it causes a 21-fold increase in unit activity of the polysome-bound enzyme (Cunningham et al., 2001
Our recent work has focused on identifying how selectivity in PMR60 targeting to substrate mRNA is determined. This process can be replicated in transfected mammalian cells, and using this we showed that endonuclease-mediated decay involves sequences on PMR60 required for targeting the ribonuclease to polysomes and ongoing translation of its substrate mRNA (Yang and Schoenberg, 2004
). This is a selective process that involves formation of an mRNP complex of PMR60 with its translating substrate mRNA. This work was facilitated by the use of a catalytically inactive form of PMR60 (PMR60°), and domain mapping experiments showed that sequences in the C-terminus are required both for polysome targeting and mRNA decay. The C-terminal polysome-targeting domain contains a consensus Src homology 2 site, and tyrosine phosphorylation at position 650 is required for PMR60 targeting to polysomes and mRNA decay (Yang et al., 2004
). This was the first demonstration of a role for tyrosine kinase signaling in mRNA decay, and our recent identification of c-Src as the responsible kinase (Peng and Schoenberg, 2007
) raises the possibility that the transforming activity of this protooncogene may in part result from increased decay of mRNAs encoding proteins that regulate cell growth.
Initial evidence linking c-Src to PMR60 came from experiments in which immunoprecipitated myc-tagged PMR60° was incubated in vitro with [γ-32
P]ATP (Peng and Schoenberg, 2007
). Tyrosine kinases commonly form a complex with their substrates and undergo autophosphorylation, and this experiment resulted in 32
P labeling of three proteins: PMR60°, c-Src, and an unidentified 90-kDa protein. Here, we identify this protein as 90-kDa heat shock protein (Hsp90), we show that this interaction is required for endonuclease-mediated mRNA decay, and we show that PMR60 is an inherently unstable protein that upon inhibition of Hsp90 is rapidly degraded by the proteasome.