PTH mRNA contains a 63 nt-long ARE-like region in its 3'-UTR that determines PTH mRNA stability [20
]. This element leads to decreased mRNA levels of reporter genes in transfected cells [20
]. The regulated binding of the PTH mRNA stabilizing proteins, AUF1 and Unr, and the destabilizing factor KSRP to this 63 element controls PTH mRNA levels in vivo in the parathyroid gland and in vitro in transfected cells [1
]. KSRP promotes rapid mRNA decay by recruiting the exoribonucleolytic complex exosome to its target mRNAs [21
]. We now show that the endoribonuclease PMR1 decreases PTH mRNA levels in transfected cells and that this involves the PTH mRNA ARE, KSRP and the exosome.
Few vertebrate mRNA endonucleases have been identified, one of which is PMR1. Since a PMR1 immunoreactive protein similar to the mammalian ortholog [24
] is expressed in the parathyroid, we asked whether this endoribonuclease may be part of the PTH mRNA decay machinery. Reagents for mammalian PMR1 are not available so we used the closely-related Xenopus protein [18
]. Over-expression of the catalytically active form of PMR1 (PMR60) decreased PTH mRNA levels in co-transfected cells and this was dependent upon the PTH mRNA 63 nt ARE. Similarly, in IVDA experiments, extracts from PMR60 over-expressing cells led to a more rapid decay of PTH mRNA with an intact ARE, when compared to extracts from mock-transfected cells. These results identify PTH mRNA and in particular the PTH mRNA ARE as a target for PMR1 in transfected cells. A PMR60 enriched fraction also specifically cleaved PTH mRNA in vitro. At least one cleavage site was identified ~70 nt from the PTH mRNA 3' end. It has been reported that PMR1 preferentially cleaves single-stranded RNAs at UG dinucleotides within albumin mRNA [25
]. The PTH mRNA 3'-UTR is an open region with little folded base pairing [20
] and contains several UG dinucleotides that may be potential targets for PMR1. Specifically, the 3' terminal region of rat PTH mRNA contains a single UG dinucleotide compatible with the production of a ~70 nt 3' PTH mRNA fragment upon digestion. Interestingly, this UG dinucleotide is part of the PTH mRNA 3'-UTR 63 nt ARE instability element that is both necessary and sufficient to confer regulation of PTH mRNA stability by changes in calcium and phosphate levels by rat parathyroid extracts and in vitro in transfected cells [19
We also show that PMR600
specifically interacts with PTH mRNA. Furthermore, PMR60 displays an unanticipated association with the exosome component Rrp4 and with KSRP. Both interactions occur in the absence of PTH mRNA. It is of interest that KSRP KH domains 3-4 that mediate KSRP-exosome association and promote ARE-containing mRNA decay [21
], are sufficient for KSRP-PMR60 association. KH domains 3 and 4 also mediate the binding of KSRP to the PTH mRNA ARE [3
]. We can hypothesize that KSRP recruits PMR1 to additional labile mRNAs and this protein-protein association would facilitate KSRP-dependent decay promoting activity.
Our results show that the effect of the endoribonuclease PMR1 on PTH mRNA levels is dependent upon the expression of exosome components and KSRP, which interacts with both the exosome and PMR1. These results suggest that PMR1, KSRP and the exosome participate in PTH mRNA decay by forming a multi-subunit degradation complex with the PTH mRNA ARE (Fig. ). Upon PMR1 cleavage, PTH mRNA decay may proceed through exosome mediated removal of the 5' fragment or of both fragments by exonucleolytic cleavage. Of interest, PMR60 over-expression prevented the increased PTH mRNA levels induced by either exosome or KSRP depletion, suggesting that PMR60 may also have an effect on PTH mRNA expression that is independent of both KSRP and the exosome.
Figure 6 Model for the role of PTH mRNA interacting proteins in PTH mRNA stability. PTH mRNA stability is regulated through the interaction of AUF1, Unr and KSRP with the PTH mRNA 3'-UTR ARE (light green) [1-3]. KSRP, the exosome and PMR1 are recruited to PTH (more ...)
Cooperation between ribonucleolytic machineries have been reported also in other systems. Knock-down of the de-capping protein Dcp2 reduces decay from the 3' end of a β-globin mRNA carrying the c-fos ARE, and knockdown of the exosome subunits PM/Scl-100 or Rrp41 reduce decay from the 5' end, indicating that the 5' and 3' decay pathways are functionally linked and that unstable mRNAs can be degraded simultaneously from both ends [7
]. Wang and Kiledjian have similarly shown that mRNA decay proceeds through a coupled 3' to 5' and 5' to 3' exoribonucleolytic pathway that involves the interaction of a sub-set of the exosome proteins with DcpS and the decapping pathway [26
]. Moreover, it was recently shown that eukaryotic exosome itself contains both exonuclease and endonuclease activity, mediated by two distinct domains of its Dis3 (Rrp44) subunit. [27
]. Our studies do not exclude the possibility of an endonuclease activity of the exosome that co-purifies with PMR60 and is distinct from PMR60 and cleaves PTH mRNA. The combination of endo and exoribonucleases in one RNA-degrading machine may offer a fundamental advantage to the cell and appears to be more widespread in nature than could be expected [28
]. Altogether, the above observations and the data presented here demonstrate that interactions of the exosome complex with other mRNA decay enzymes facilitate and coordinate mRNA decay, both endo- and exonucleolytically.