Mouse RING finger protein Rnf133 is a testis-specific endoplasmic reticulum-associated E3 ubiquitin ligase

Abstract

Spermatogenesis, the process by which sperms are generated within the male gonads, involves a number of events that occur only in the testis. Examples include the nucleus condensation as a result of histone sequential replacement by transition proteins 1, 2 and protamin, the formation of sperm-specific organelles such as acrosomes and sperm tails, and the shedding of majority of the cytoplasm as residual bodies. To support the accomplishment of these events, it is hypothesized that a large number of testis-specific genes need to be expressed, which is consistent with the estimation that about 4% of the genes in the mouse genome are specifically expressed in the testis [1]. Identification of these genes and their biological functions will not only help us to elucidate the mechanisms of spermatogenesis but also provide potential targets for novel non-hormonal contraceptive drugs [2].In this study, we identified a potential testis-specific gene named Rnf133 (NCBI: Mm.436547). It belongs to the RING finger protein (RNF) family, whose members are very divergent in terms of their sequences and functions except for sharing a RING domain (CX2CX(9-39)CX(1-3)HX(2-3)C/HX2CX(4-48)CX2C) [3]. A number of RNFs have been reported to be expressed in the testis, but their functions are poorly understood [4]. Multiple sequence alignment of Rnf133 proteins from five mammalian species (Mus musculus, Homo sapiens, Pan troglodytes, Rattus norvegicus, Macaca mulatta) indicated that they are highly conserved (~70% identity among all species, Supplementary information, Figure S1). All these proteins possess a C3H2C3 RING finger domain, a protease-associated (PA) domain, a signal peptide (SP) and a transmembrane (TM) domain. These features are typical of the GREUL (for Goliath Related E3 Ubiquitin Ligase) proteins, a group of RNFs with some of its members being identified as E3 ubiquitin ligases [5].Northern blotting analysis of 14 adult mouse tissues indicated that Rnf133 was detected only in the testis ( Figure 1A). RT-PCR results indicated that Rnf133 mRNA started to appear in the testis of day 21 mice and increased dramatically from day 28 and thereafter ( Figure 1B). As round spermatids appear by day 21 during spermatogenesis in mice, Rnf133 seemed to be expressed by round spermatids. This was supported by the results of in situ hybridization in which strong signals were detected in the adluminal regions of the seminiferous tubules where haploid germ cells are located ( Figure 1C). The interesting observation that signal was not observed in all of the seminiferous tubules strongly implied stage-specific expression of Rnf133 during spermatogenesis. Indeed, immunohistochemical staining using a rabbit polyclonal antiserum developed against the region from aa 209 to aa 382 (see the Supplementary information, Materials and Methods for procedures of the production of the antiserum and Figure S2 for its specificity characterization) revealed that Rnf133 was present in the cytoplasm of the step 14-16 elongated spermatids in the stage II-VI ...

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 4: Intercellular bridges, mitochondria, nuclear envelope, apoptosis, ubiquitination, membrane/voltage‐gated channels, methylation/acetylation, and transcription factors

“…All of these features are common of the GREUL (Goliath Related E3 Ubiquitin Ligase) proteins, a group of RNFs with some members identified as E3 ubiquitin ligases (Borchers et al, 2002). RNF133 is specifically expressed in the testis localizing to the endoplasmic reticulum of step 14-16 elongating spermatids, where it functions as an E3 ubiquitin ligase (Nian et al, 2008).…”

Identification of a novel human testicular interstitial gene, RNF148, and its expression regulated by histone deacetylases

“…The aa sequence of the conserved C3H2C3 RING-H2 finger domain has a high identity profile with that of human E3 ubiquitin ligases RNF128 (Anandasabapathy et al, 2003), RNF130 (Guais et al, 2006), and mouse Rnf133 (Nian et al, 2008) (Figure 5). Of these, RNF128 regulates T-cell tolerance and induces T-cell anergy through the ubiquitination activity of its RING finger domain (Nurieva et al, 2010;Whiting et al, 2011).…”

“…Of these, RNF128 regulates T-cell tolerance and induces T-cell anergy through the ubiquitination activity of its RING finger domain (Nurieva et al, 2010;Whiting et al, 2011). Mouse testis-specific protein Rnf133 is mainly present in the cytoplasm of elongated spermatids and plays a role in sperm maturation through an endoplasmic reticulum-associated degradation pathway (Nian et al, 2008). Because RNF148 has primary structures that parallel those of E3 ubiquitin ligases, we also constructed several mutant transcripts of RNF148 with Flag-and HA-tag vectors to test whether RNF148 also functioned similarly as an E3 ubiquitin ligase.…”

The Role of the Transmembrane RING Finger Proteins in Cellular and Organelle Function

“…Almost 4% of the genes in the mouse genome are estimated to be male-germ-cell specific [128], and a number of testis-specific RNF proteins have been identified which are thought to play a role in spermatogenesis and fertilization. RNF133, a member of the PA-TM-RING family, is an E3 enzyme that is highly and specifically expressed in male germ cells undergoing spermiogenesis (spermatids) [129]. RNF133 is localized to the ER in the immortalized mouse spermatocyte cell line GC-2 and catalyzes self-ubiquitination in 293T cells, suggesting that RNF133 is involved in ER protein quality control during spermatogenesis.…”