KSRP-PMR1-exosome association determines parathyroid hormone mRNA levels and stability in transfected cells
© Nechama et al; licensee BioMed Central Ltd. 2009
Received: 21 January 2009
Accepted: 23 September 2009
Published: 23 September 2009
Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally through the binding of the trans- acting proteins AU rich binding factor 1 (AUF1), Upstream of N-ras (Unr) and KH-type splicing regulatory protein (KSRP) to an AU rich element (ARE) in PTH mRNA 3'-UTR. AUF1 and Unr stabilize PTH mRNA while KSRP, recruiting the exoribonucleolytic complex exosome, promotes PTH mRNA decay.
PTH mRNA is cleaved by the endoribonuclease polysomal ribonuclease 1 (PMR1) in an ARE-dependent manner. Moreover, PMR1 co-immunoprecipitates with PTH mRNA, the exosome and KSRP. Knock-down of either exosome components or KSRP by siRNAs prevents PMR1-mediated cleavage of PTH mRNA.
PTH mRNA is a target for the endonuclease PMR1. The PMR1 mediated decrease in PTH mRNA levels involves the PTH mRNA 3'-UTR ARE, KSRP and the exosome. This represents an unanticipated mechanism by which the decay of an ARE-containing mRNA is facilitated by KSRP and is dependent on both the exosome and an endoribonuclease.
Parathyroid hormone (PTH) regulates serum calcium and phosphate levels and bone strength. Serum calcium and phosphate concentrations, in turn, control PTH gene expression post-transcriptionally through regulated binding of the trans- acting proteins AU rich binding factor 1 (AUF1), Upstream of N-ras (Unr) and KH-type splicing regulatory protein (KSRP) to a type III AU rich element (ARE) in PTH mRNA 3'-UTR [1–3]. AUF1 and Unr stabilize PTH mRNA both in an in-vitro degradation assays (IVDA), using parathyroid extracts, and in intact cells. We have recently shown that the mRNA decay promoting protein KSRP decreases PTH mRNA stability and steady-state levels through the PTH mRNA ARE . Both KSRP and AUF1 bind to PTH mRNA in vitro and in intact parathyroid glands . In the parathyroid, the interaction of PTH mRNA with KSRP and AUF1 is regulated by changes in serum calcium and phosphate. Calcium depletion increases the association of AUF1 with the PTH mRNA ARE and decreases KSRP binding to the ARE resulting in mRNA stabilization. These interactions are reversed by phosphate depletion where PTH mRNA is destabilized . In tranfected cells, over-expression of KSRP destabilizes the PTH mRNA and this is mediated by the PTH mRNA ARE. KSRP-PTH mRNA interaction is prevented by over-expression of AUF1 p45 isoform. Over-expression of AUF1 p45 also attenuates the KSRP-mediated destabilization of PTH mRNA in transfected cells . The peptidyl-prolyl isomerase Pin1 is also a PTH mRNA destabilizing protein. Pin1 mediates its effects via interaction with KSRP, which leads to KSRP dephosphorylation and activation . The regulated interactions of KSRP and AUF1 with the PTH mRNA ARE determine its half life in vivo and in vitro.
AREs are destabilizing elements located in the 3'UTRs of many inherently labile mRNAs . AREs are targets for trans-acting proteins regulating mRNA localization, stability and translation . Upon deadenylation, ARE-containing mRNAs are degraded in either a 3' to 5' or a 5' to 3' direction by two distinct exoribonucleolytic pathways mediated by the exosome and XrnI, respectively . It has been recently demonstrated that these two pathways are functionally linked [7, 8]. KSRP recruits the multiprotein 3'-5'exoribonuclease complex, exosome to target mRNAs. The central part of KSRP contains four adjacent KH domains that are required for its interaction with the decay-promoting machinery and with ARE containing mRNAs . AU rich binding factor 1 (AUF1) promotes either decay or stabilization, depending on the mRNA and cell type .
In addition, a number of mRNAs are targeted by endonucleases that initiate decay by cleaving within the body of the mRNA while it is actively engaged by translating ribosomes. Three mRNA endonucleases have been linked to specific decay pathways; polysomal ribonuclease 1 (PMR1) , G3BP  and IRE-1 . In Xenopus Laevis hepatocytes, many mRNAs are destabilized by estrogen through the activation of PMR1 . PMR1 forms a selective complex with its substrate mRNA to initiate decay by cleaving within the mRNA . Xenopus (x) PMR1 is a member of the peroxidase gene family and is synthesized as a 80-kDa precursor (PMR80) that is processed to the active 60-kDa form (PMR60) . The ability of PMR1 to target polysomes and activate mRNA decay depends on tyrosine phosphorylation at position 650 in the C-terminus of PMR60 by c-Src [16, 17].
Here we show that PTH mRNA is a substrate for PMR1 in vitro and in transfected cells. The PTH mRNA 3'-UTR ARE is required for PMR60-dependent PTH mRNA destabilization. PMR60 co-immunoprecipitates with PTH mRNA, the exosome and KSRP. Surprisingly, siRNA mediated knock-down of either exosome components or KSRP reduces the PMR1-mediated PTH mRNA decay in intact cells. We suggest that KSRP recruits a degradation complex, comprising both endo- and exo-ribonucleases to PTH mRNA, thus controlling its mRNA half-life.
PMR60 decreases PTH mRNA and protein levels in transfected HEK293 cells by promoting ARE-dependent PTH mRNA decay
PTH gene expression is largely controlled by an ARE in the 3'- UTR of PTH mRNA [3, 19]. To determine if the regulation of PTH mRNA levels by PMR60 is exerted via the PTH mRNA ARE, We used a rPTH expression plasmid with an internal deletion of the PTH mRNA 3'-UTR ARE. In contrast to the full-length PTH mRNA, co-tranfected PMR60 had no effect on the mutated PTH mRNA (Fig. 1E). Therefore, the decrease in PTH mRNA levels by PMR60 is dependent on an intact ARE. We then used a GH reporter gene containing the rat PTH 63 nt ARE (GH63) . Transfected PMR60 reduced GH63 mRNA levels but had no effect on wild type GH or endogenous β-actin mRNA levels (Fig. 1F-G). These results demonstrate that PMR60 specifically reduces steady-state PTH mRNA levels through the PTH mRNA ARE which is both necessary and sufficient for this effect.
PMR60 associates with PTH mRNA in transfected cells and cleaves PTH mRNA in vitro
We then studied whether PTH mRNA is a substrate for partially purified PMR60 in vitro. We used the catalytically active form of TAP and Myc-tagged PMR1 (PMR60) , recovered by IgG-sepharose affinity purification from transfected cultured cells. PMR60 cleaved in vitro transcribed uniformly labeled PTH mRNA but not GH mRNA (Fig. 3B, bottom panel).
PMR60 also cleaved a 3'-end labeled rat PTH mRNA and generated a single 3'-end labeled cleavage product of approximately 70 nt, that was detected by urea-PAGE (Fig. 3C). A control GH transcript was not cleaved (data not shown), as with uniformly labeled GH mRNA (Fig. 3B). Our results suggest that PTH mRNA is a target for PMR60 in vitro and that this endoribonuclease cleaves PTH RNA at a site located approximately 70 nt from its 3' end.
PMR600 associates with the exosome component Rrp4 and with KSRP
We next studied the association of PMR60 with the exosome in Cos1 cells. PMR60 over-expression has a similar effect to decrease co-transfected PTH mRNA levels in these cells (not shown). Cos-1 cells were co-transfected with plasmids expressing TAP and Myc-tagged PMR600 and either Flag-GFP or Flag-Rrp4, one of the core exosome subunits. Cytoplasmic extracts were immunoprecipitated by anti-Flag antibody. Anti-Myc immunoblotting of the immuno-complexes revealed that PMR600 was specifically recovered by Rrp4 but not by GFP (Fig. 4C upper panel, bound). Altogether, our findings indicate that PMR60 associates with both KSRP and the exosome.
KSRP, PMR60, and the exosome are components of the PTH mRNA decay machinery
We also studied the ability of PMR1 to reduce PTH mRNA levels in KSRP depleted cells. KSRP was knocked down by specific siRNAs in cells expressing PTH and either PMR60 or control plasmid. KSRP knock-down (~60%, Fig. 5D) increased PTH mRNA levels (Fig. 5E and 5F) as we have previously reported . Interestingly, KSRP knock-down partially prevented the PMR60-induced decrease in PTH mRNA levels (Fig. 5E and 5F). Similar results were obtained using an additional set of siRNAs targeting a different sequence of KSRP mRNA (data not shown). Altogether our results indicate that the exosome and KSRP participate in the PMR60-dependent decrease in PTH mRNA levels in cultured cells.
PTH mRNA contains a 63 nt-long ARE-like region in its 3'-UTR that determines PTH mRNA stability [20, 22]. This element leads to decreased mRNA levels of reporter genes in transfected cells [20, 23]. 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 [1–3]. KSRP promotes rapid mRNA decay by recruiting the exoribonucleolytic complex exosome to its target mRNAs . 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  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 . 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 . The PTH mRNA 3'-UTR is an open region with little folded base pairing  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, 20, 22].
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 , are sufficient for KSRP-PMR60 association. KH domains 3 and 4 also mediate the binding of KSRP to the PTH mRNA ARE . We can hypothesize that KSRP recruits PMR1 to additional labile mRNAs and this protein-protein association would facilitate KSRP-dependent decay promoting activity.
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, 8]. 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 . 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, 28]. 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 . 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.
PTH mRNA is a target for the endonuclease PMR1. The PMR1 mediated decrease in PTH mRNA levels involves the PTH mRNA 3'-UTR ARE, KSRP and the exosome. Our findings suggest an unanticipated mechanism by which KSRP and the exosome regulate the half-life of a target mRNA by facilitating its endoribonucleolytic cleavage.
For IVDA post-mitochondrial extracts were prepared. Cultured cells were incubated on ice for 10 min in an extraction buffer containing 0.25 M sucrose, 30 mM Tris HCl pH 7.5, 2 mM DTT, and a protease inhibitor mix. Samples were homogenized and the supernatant cleared by centrifugation at 15,000 g for 15 min (4°C). For Western blots, extracts from cultured cells were prepared using RIPA buffer containing 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitors. Extracts were stored in aliquots at -80°C.
Cell cultures and transfections
HEK293 cells were transiently transfected with different expression plasmids using a Ca-P transfection kit (Sigma, St Louis MO). siRNA oligonucleotides were transiently co-transfected with expression plasmids using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA).
PMR60 purification and in vitro activity assay
Xenopus (x) PMR60 was purified as previously described . Briefly, an expression plasmid for active form of xPMR1 (Myc-PMR60-TAP) was transfected into HEK293 cells and after 48 h PMR60 was recovered from cell lysate by IgG-Sepharose 6 Fast Flow (Amersham, Little Chalfont UK), followed by Tev protease cleavage of the TAP tag. Purified PMR60 was incubated with uniformly labeled PTH mRNA transcript for 1 h at room temperature in a reaction buffer containing 30 mM Tris HCl pH 7.5, 1 mM DTT, 2 mM MgCl2 and 75 mM KCl.
PTH mRNA recovery by PMR600
HEK293 cells were transiently co-transfected with the catalytically inactive PMR form, xPMR600, and expression plasmids for either the hPTH gene or luciferase cDNA. xPMR600 containing complexes were recovered from cell extracts on IgG-Sepharose and cleavage with Tev protease. RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) and reverse transcribed using Super-Script II Reverse Transcriptase. cDNA was analyzed by semi quantitative PCR with γ [32P] ATP.
Cos-1 cells were co-transfected with constructs for xPMR600 and either Flag-GFP or Flag-Rrp4. Cytoplasmic extracts were applied to monoclonal anti-Flag M2 agarose beads (Sigma, St Louis MO), eluted and analyzed by SDS-PAGE and Western blots.
HEK293 cells were co-transfected with expression plasmids for either TAP and Myc-tagged xPMR600 or GFP-TAP, with or without expression plasmid for Flag-tagged KSRP or truncated KSRP containing the different KH domains. GFP and PMR60 were recovered by IgG-Sepharose 6 Fast Flow, digested by Tev protease, and analyzed by immunoblots for endogenous KSRP using an anti KSRP antibody, or for the transiently transfected Flag-tagged KSRP using anti Flag antibody. In some experiments 40 μg/ml of RNase A and 25 μg/ml of RNase T1 were added to the protein extracts to determine if interactions were RNA dependent. After incubated at room temperature for 20 min. proteins were recovered and analyzed by immunoblots as above.
Previously published siRNAs targeting Rrp46 :5'-CAAGGCCACACUCGAAGUG-3', Rrp40: 5'-GGAGACCAUGUGAUUGGCA-3' , KSRP: 5'-AAGATCAACCGGAGAGCAAGA-3'  and an additional set of commercial, siRNAs for KSRP (sequence not available) and the control CAT: 5' r(GACGGUGAGCUGGUGAUAU)d(TT)-3' or TTCTCCGAACGAACGTGTCACGT-3', were all synthesized by QIAGEN (Hilden, Germany).
RNA was extracted with Tri-Reagent (Molecular Research Center, Cincinatti, OH) and analyzed as previously described .
RNA was reverse transcribed with random hexamer primers using a Maxime RT premix kit (iNtRON Biotechnology, Gyeonggi-do, Korea), and analyzed by real-time quantitative polymerase chain reaction (qPCR) using ABI Prism 7901 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and SYBR Green ROX Mix (ABgene, Epsom, UK).
PCR primers for qRT-PCR
Rat PTH primers were: 5'-TTGTCTCCTTACCCAGGCAGAT-3' and 5'-TTTGCCCAGGTTGTGCATAA-3'. Primers for β-actin were: 5'-CAGGCATTGCTGACAGGATG-3' and 5'-CTCAGGAGGAGCAATGATCTTGAT-3'.
Proteins were analyzed by SDS PAGE immunoblots as previously described .
RNA transcription and labeling
Uniformly α[32P] UTP labeled polyadenylated RNAs for the full-length PTH mRNA, a PTH mRNA with an internal deletion of the ARE or GH mRNA were transcribed in vitro as previously described [3, 19]. For 3'-end labeled PTH mRNA transcripts, the rat PTH cDNA containing plasmid was linearized with BclI and unlabeled RNA transcribed and extracted using Tri Reagent. RNA was then annealed to a primer homologous to the 3'-end of the transcript, leaving a 2 base 5' overhang. The sequence of the primer was 5'-TGATTAAACTTT-3'. The 3'-end of the transcript was then labeled using 4 U of Klenow enzyme, 2 μM dNTP and α[32P] dCTP by incubation at 37°C for 2 h. All labeled transcripts were purified using mini Quick Spin RNA Columns (Roche, Mannheim, Germany).
In Vitro Degradation Assays (IVDA)
Radiolabeled transcripts were incubated with 50 μg proteins of post mitochondrial cell extracts in a reaction buffer containing 3 mM Tris HCl, pH 7.5, 2 mM MgCl2, 3 mM NaCl, 10 mM ATP and 80 units/ml RNasin and analyzed as described . At timed intervals samples were removed separated on formaldehyde agarose gels or urea SDS PAGE and analyzed by autoradiography.
Rat PTH cDNA was cloned in either pcDNA3  for transient transfections or in pBluescript II KS  for in vitro transcription for IVDAs. The pBluescript II KS plasmid containing the full-length rat PTH cDNA including a stretch of ~150 dT nucleotides that by in vitro transcription produced a poly A tail was used. An internal BsaI-BclI fragment (~80 bp) was removed by partial restriction enzyme digestion of the pBluescript II KS-PTH cDNA plasmid, followed by ligation to produce a plasmid without the ARE . The human PTH gene was in pcDNA3 . The firefly luciferase plasmid was in pGL3 (Promega, Madison WI). The GH gene expression plasmid was kindly provided by O. Meyuhas, Hadassah Medical School, Jerusalem, Israel) . The GH-PTH mRNA 63 nt plasmid (GH63) was previously described  and contained the 63 nt rat PTH mRNA ARE cloned between the 3' end of the GH mRNA coding sequence and the GH mRNA 3'-UTR. The corresponding GH cDNAs cloned into pBluescript II KS were used for in vitro transcription. Plasmids coding for the active (PMR60) or inactive (PMR600) forms of Xenopus PMR1 contained a N terminal Myc tag and a C terminal TAP tag and were in pcDNA3 . KSRP in pcDNA3 contained either Flag-tagged full-length KSRP or different KSRP KH domains . Empty control vectors (pcDNA3 and pSG5) were used as indicated.
Anti-KSRP was previously described . The anti-Flag, anti α-tubulin, and anti-GFP were from Sigma (St Louis MO). Anti-Myc was from Cell Signaling (Boston, MA, USA)
Serum PTH measurements
Serum rat/human PTH was measured using the rat/human intact PTH ELISA Kits (Immunotopics, San Clemente, California, USA).
Values are reported as mean ± SEM unless stated otherwise. A 2-tailed p value was considered significant when less than 0.05.
AU rich element
AU rich binding factor 1
human embryonic kidney
in vitro degradation assay
KH-type splicing regulatory protein
- qRT PCR:
quantitative reverse trasnscriptase polymerase chain reaction
small interfering RNAs
Upstream of N-ras
This work was supported in part by grants from the Israel Academy of Sciences, an ASBMR bridging fund and the Chief Scientist of the Ministry of Health (TNM), by a ISS grant # 526D/39 (PB) a CIPE Regione Liguria (RG) and a NIH grant R01 GM38277 (DRS).
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