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First published online 18 December 2002
doi: 10.1242/jcs.00261

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hRUL138, a novel human RNA-binding RING-H2 ubiquitin-protein ligase

University Hospital Freiburg, Department of Internal Medicine II, Molecular Biology, Hugstetter Str. 55, D-79106 Freiburg, Germany

View larger version (29K): [in a new window]   Fig. 1. Schematic representation of the hRUL138 ORF and protein. (A) cDNAs identified in this study. The line on the top represents the complete hRUL138 ORF, the bars below indicate cDNA sequences. Numbers are nt positions relative to the hRUL138 ORF. Non-translated regions are depicted by hatched bars and regions encoding identical amino-acid sequences as in hRUL138 by dark grey bars. Light grey stippled bars correspond to identical nucleotide sequences which are, however, out of frame owing to small, splicing-related deletions (shown as ) that lead to early translational stops (*); (A)n denotes a poly-A tail. White bars indicate introns that have not been spliced out in the corresponding cDNAs. NIII is the cDNA initially isolated by Northwestern screening, the other cDNAs were identified by molecular hybridization using an NIII derived DNA probe (black line above NIII). (B) Predicted motifs within the hRUL138 primary sequence. Numbers are amino acid positions. The lysine-rich (K-rich) motif and the RING-H2 domain are indicated by black boxes; the RING-H2 sequence is shown below with the conserved Cys and His residues highlighted in bold face. The prediction for the coiled-coil motif is strong between amino acids 794 to 852 and extends with weaker scores as indicated by the hatched bars. The weakly and variably predicted putative transmembrane regions are denoted by TM?

View larger version (31K): [in a new window]   Fig. 2. Mapping of the RNA-binding region in hRUL138. (A) Deletion mapping. Crude lysates from bacteria expressing MBP-fused hRUL138 fragments truncated at the indicated positions (scheme above the gels; stippled bars outline hRUL138 parts not present) were tested for binding to a DIG-labeled HBV--containing RNA probe by Northwestern blotting. Bound RNA was visualized using anti-DIG antibody followed by chemiluminescent detection (left panel). The band at about 50 kDa is most likely a proteolytic hRUL138 fragment. Equal loading was confirmed by Coomassie blue staining of the SDS-PAGE gel (right panel) used to generate the blot. (B) Mutagenesis of the K-rich motif. Internal hRUL fragments amino acids 510 to 878 containing the authentic K-rich motif or the variant K4- (amino acids 662 to 666=SGSTA) and amino acids 832 to 1176 were expressed in E. coli as fusions with the pET30 encoded linker, and processed as in A. For Northwestern analysis the blot was incubated with DIG-labeled HBV RNA (left panel); the bands below the 46 kDa marker position are probably degradation products. To control for loading, the same blot was reprobed with an antiserum recognizing the His-tag containing pET30 linker. (C) In-solution RNA binding. The same proteins as in B, except that a fragment comprising amino acids 1 to 205 was used as negative control, were incubated with 32P-labeled HBV RNA, immobilized on Ni-NTA beads, and the radioactivity remaining on the beads was determined by scintillation counting. Results are shown as a percentage of the cpm measured for the authentic 510-878 fragment.

View larger version (59K): [in a new window]   Fig. 3. RNA-binding specificity of hRUL138. The same wild-type and K4- variant hRUL138 fragments as in Fig. 2 were expressed as pET30 linker fusions and processed as before for Northwestern blotting, except that RNA probes derived from a different part of the HBV genome (panel HBV-RT) or an unrelated RNA derived from the alfalfa mosaic virus leader RNA (panel AMV) were used as probes. Loading was controlled by reprobing the blots with an anti-His antibody. Note that weak but detectable signals were also observed in the Northwestern blot lanes containing the K4- mutant proteins.

View larger version (44K): [in a new window]   Fig. 4. Self-ubiquitylation of hRUL138. (A) Polyubiquitylation of full-length hRUL138. hRUL138 was in vitro translated in the presence of 35S-Met and incubated with ATP and E1 plus ubiquitin (Ub) and UbcH5 or UbcH7 as indicated, and the reaction products were analyzed by SDS-PAGE and autoradiography. The borders of the stacking gel are indicated; material accumulating at the upper edge of the stacking gel is marked poly-Ub RUL. (B) Effect of methyl ubiquitin. hRUL138, and the RING containing fragment 681-1208 were in vitro translated and processed as in A, except that methyl ubiquitin (MeUb) was used in two reactions. Both proteins were efficiently modified except that the reaction products were smaller (smear extending to the top of the separating gel; lanes 9 and 12). (C) Requirement for an intact RING-H2 domain. RUL681-1208 and its C1187S mutant were in vitro translated and subjected to ubiquitylation assays as in A.

View larger version (45K): [in a new window]   Fig. 5. Trans-ubiquitylation by hRUL138. MBP-RUL681-1208-FLAG and His-RUL681-1128-His, lacking the RING domain (schematically depicted on the top; RING+ and RING-, respectively) were co-expressed in E. coli and the material immobilized on amylose resin was subjected to ubiquitylation assays with methyl ubiquitin. Alternatively, the separately expressed proteins were analyzed individually or in a mixture (lanes mix). Reactions were separated by SDS-PAGE, blotted and incubated with either anti-His antibody (left panel; RING- proteins) or with anti-FLAG antibody (right panel; RING+ proteins). The asterisk denotes a probable dimer of the RING- protein.

View larger version (62K): [in a new window]   Fig. 6. Upon proteasome inhibition, hRUL138 steady-state levels in intact cells increase in a RING-dependent fashion. Transiently transfected Huh7 cells expressing GFP fusions of hRUL138 or the RING-deleted variant RUL1-1128 were treated for 6 hours with the proteasome inhibitor MG132, or DMSO alone, and equal aliquots from total cell lysates were analyzed by western blotting using a monoclonal anti-GFP antibody (left panel). The stabilizing effect of MG132 was also observed when the FLAG epitope instead of GFP was fused hRUL138; no signal at the corresponding position was detectable in nontransfected MG132-treated Huh7 cells (right panel).

View larger version (113K): [in a new window]   Fig. 7. Tissue-specific expression of hRUL138-related mRNAs. A multiple tissue Northern blot containing poly-A+ RNA from the indicated human tissues was probed with a 32P-labeled hRUL138 DNA (nt positions 1497-1998 of the hRUL ORF) and analyzed by phosphorimaging. For control, the same blot was reprobed with an actin probe (lower panel). Numbers on the left show the positions of RNA size markers (in kb); the major hRUL transcripts are indicated by the arrowheads on the right.

View larger version (72K): [in a new window]   Fig. 8. Intracellular localization of hRUL138. Huh7 cells were transfected with expression constructs for the GFP fusion proteins indicated on the top of each panel. For colocalization studies, the indicated hRUL-GFP construct was co-transfected with plasmid pRFP2-ER, encoding an ER-targeted red fluorescent protein. Unfixed cells were observed by confocal laser scanning microscopy, except for the TOTO-3 counterstaining in panel B; there GFP was detected indirectly using mouse anti-GFP antibodies and a fluorescently labeled anti-mouse IgG antibody. (A) Localized distribution of hRUL138-GFP versus even distribution of GFP alone. (B) Extranuclear staining by hRUL1-1128-GFP versus blue nuclear staining by TOTO-3. (C) Coexpression of hRUL1-1128-GFP and RFP2-ER. (D) Coexpression of hRUL510-878-GFP and RFP2-ER.