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

Abstract

Cellular as well as viral RNAs are usually found complexed with proteins. In an attempt to identify proteins that interact with transcripts of hepatitis B virus (HBV), a DNA virus that replicates through reverse transcription, a partial cDNA was isolated from a human cDNA expression library whose gene product bound to an HBV-derived RNA. Using an overlapping clone from a molecular hybridization screen a full-length cDNA was assembled. It contained a large open reading frame for a 1208 amino-acid protein of 138 kDa identical to the hypothetical product of the KIAA0675 clone. Closely related sequences are present in mouse cDNA libraries but not in the genomes of lower organisms. The protein sequence contained no known RNA-binding domain and, apart from a probable coiled-coil domain, the only significant homology involved a complete RING-H2 motif. This suggested that the protein might be a novel RNA-binding RING-dependent ubiquitin-protein ligase or E3 enzyme. A motif critical for RNA binding was experimentally mapped to a central Lys-rich region. Binding specificity is either broad or the protein has as yet unknown physiological targets; hence, at present, a potential importance for HBV biology remains open. The RING-H2 domain was functional in and essential for self- and trans-ubiquitylation in vitro and for proteasome-mediated turnover of the protein in vivo. We therefore termed it hRUL138 for human RNA-binding ubiquitin ligase of 138 kDa. hRUL138 mRNAs are expressed at low levels in most tissues. GFP-tagged hRUL138 derivatives were found associated with cytoplasmic structures, possibly the ER, but excluded from the nucleus. The combined presence of RNA binding and E3 activity in hRUL138 raises the possibility that both are mechanistically linked.

Scaffold function of long non-coding RNA HOTAIR in protein ubiquitination

“…S1b). The latter group revealed that two E3 ubiquitin ligases with RNA-binding domains, Mex3b (a RING- and KH domain-containing E3 ubiquitin ligase 16 ) and Dzip3 (a RING- and KKKTK domain-containing E3 ubiquitin ligase 17 ) selectively interacted with HOTAIR (Supplementary Fig. S1b).…”

“…As shown in Fig. 6a, a mutant Dzip3 (bearing a point mutation in the RING domain, essential for ubiquitination) expressed in HeLa cells from plasmid pTRUF-hRUL138(C1187S) was unable to ubiquitinate Ataxin-1 (top) or promote its degradation (bottom), as compared with the effect of expressing the wild-type counterpart pTRUF-hRUL138(WT) 17 . In addition, ectopic expression of a mutant Ataxin-1(82Q) that cannot bind RNA 21 indeed did not interact with HOTAIR (Fig.…”

Hepatitis B virus replication

“…Direct screens identified a 65 kDa nuclear protein [84] of unknown sequence and, more recently, a novel large RNA-binding ubiquitin ligase, hRUL138 [85] , as potential cellular ε RNA interaction partners. However, their roles in the viral life-cycle are not known.…”

RNA-binding E3 ubiquitin ligases: novel players in nucleic acid regulation

“…The hRUL138 (human RNA-binding ubiquitin ligase 138) protein was initially discovered in a screen for human proteins which bind RNA HBV-ε-derived probes [31]. This protein encodes a RING-H2 type domain and, in vitro, can autoubiquitinate, as well as transubiquitinate targets.…”

“…This protein encodes a RING-H2 type domain and, in vitro, can autoubiquitinate, as well as transubiquitinate targets. In the absence of a known RRM within the hRUL138 sequence, Kreft and Nassal [31] used deletion mapping and Northwestern blotting to identify a novel KKKTK (Krich) region required for RNA binding. Furthermore, GFP (green fluorescent protein)-tagged hRUL138 is expressed in cytosolic punctate structures probably resembling stress granules.…”