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.