Ubiquitin ligase MKRN1 modulates telomere length homeostasis through a proteolysis of hTERT

Abstract

Telomere homeostasis is regulated by telomerase and a collection of associated proteins. Telomerase is, in turn, regulated by post-translational modifications of the ratelimiting catalytic subunit hTERT. Here we show that disruption of Hsp90 by geldanamycin promotes efficient ubiquitination and proteasome-mediated degradation of hTERT. Furthermore, we have used the yeast two-hybrid method to identify a novel RING finger gene (MKRN1) encoding an E3 ligase that mediates ubiquitination of hTERT. Overexpression of MKRN1 in telomerase-positive cells promotes the degradation of hTERT and decreases telomerase activity and subsequently telomere length. Our data suggest that MKRN1 plays an important role in modulating telomere length homeostasis through a dynamic balance involving hTERT protein stability.

Human Telomerase Exists in Two Distinct Active Complexes In Vivo

“…This property is evidently due to proteolytic degradation via proteasome pathway(s), since the proteasome inhibitors prevented the degradation of endogenous hTERT and FLAG-hTERT in the complex of 380-400 kDa. Hsp90 functions in a variety of aspects (34)(35)(36), and Hsp90 inhibitors prevent the degradation of some client proteins, and facilitate the degradation of other client proteins (22,37). With telomerase, Hsp90 may function in a way distinct from its role as chaperone that serves premature state of client proteins into mature proteins.…”

“…Heat shock protein 90 (Hsp90), a molecular chaperone, has been reported to be functionally associated with hTERT, and both Hsp90 and p23 are required for efficient telomerase assembly in vitro and in vivo, although both of these proteins, as well as other chaperones, are associated with hTERT in its unassembled or inactive form (20,21). The Hsp90 inhibitor, geldanamycin (GA), has been shown to prevent the assembly of active telomerase, as well as to promote ubiquitination and the proteasome-mediated degradation of hTERT (20,22). These reports clearly demonstrate the involvement of Hsp90 in telomerase assembly and function.…”

The putatively functional Mkrn1-p1 pseudogene is neither expressed nor imprinted, nor does it regulate its source gene in trans

“…If we are to determine the developmental function of Mkrn1 in mice, it may be necessary to concurrently functionally ablate the paralogous Mkrn2 (17) and Mkrn3 (18) genes; a caveat is that other genes in the Mkrn1 biological pathway(s) might compensate for the constitutional loss of Mkrn1 function. Makorins may have an immune function, given that an ancestral Mkrn gene was captured and integrated into a progenitor poxvirus genome (29), retaining portions encoding the RING zinc-finger E3 ubiquitin ligase domain (21)(22)(23) and in some cases also the putative Mkrn1-RNA binding zinc-finger motifs (16,19,29) to form a key virulence gene (30). Finally, because Mkrn1-p1 is not transcribed and cannot regulate Mkrn1 in trans, and because a reduction in the amount of Mkrn1 transcripts does not cause the sxl transgenic phenotype, the unconventional Mkrn1-p1 and Mkrn1 microinjection rescues of the original sxl transgenic phenotype (6) were likely an artifact of incorrect genotyping, the nonspecificity of the openeye phenotype (see Methods), and͞or other experimental error.…”

“…4A). RRB087 Mkrn1 gene-trapped transcripts lack exons 4-8, and the encoded proteins lack four of the Makorin family zinc-finger motifs, including the essential RING E3 ubiquitin ligase domain (16,17,(21)(22)(23). Therefore, the extremely truncated Makorin-1-␤-geo fusion derivative (Fig.…”

“…The Makorin (gene symbol Mkrn) gene family encodes proteins having a characteristic zinc-finger composition (16)(17)(18)(19) with two to four C3H zinc-fingers that may confer RNA-binding (20), a novel C2H2C putative zinc-finger of unknown function, and a C3HC4 RING zinc-finger that allows Makorins to function as E3 ubiquitin ligases that ubiquitinate specific target proteins (21)(22)(23). Makorin orthologs are found in all metazoans, and vertebrates have two distinct intron-containing Mkrn paralogs, Mkrn1 and Mkrn2, which arose by gene duplication Ͼ400 million years ago (16,17).…”