To identify RPS23RG1 homolog(s), we used the 141 amino acid-long mouse RPS23RG1 protein sequence to blast GenBank protein database and found another three mouse proteins with very high homology to RPS23RG1: EG381438 (Gene ID 381438, identities = 116/117, 99%), LOC100040998 (Gene ID 100040998, identities = 137/139, 98%) and LOC100039346 (Gene ID 100039346, identities = 126/134, 94%), of which the latter two are predicted by automated computational analysis. Hence, we designated their encoding genes as Rps23rg2 (protein RPS23RG2 for EG381438), Rps23rg3 (RPS23RG3 for LOC100040998) and Rps23rg4 (RPS23RG4 for LOC100039346), respectively (Fig. A). RPS23RG1 has a predicted transmembrane domain (Fig. A) and previously we found that RPS23RG1 is a type Ib transmembrane protein that has a normal type I transmembrane protein orientation but no cleavable signal peptides. Here we found that RPS23RG2–RPS23RG4 also have the predicted transmembrane domain (Fig. A), suggesting that they are also type Ib transmembrane proteins. Blast with the mouse RPS23RG1 protein sequence also identified a predicted hypothetical protein LOC738579 (Gene ID 738579, identities = 50/74, 67%) with a relatively high homology, but lacking the predicted transmembrane domain from chimpanzees (Pan troglodytes) (Fig. A), which we designated Rps23rg5 (and the protein as RPS23RG5). Moreover, the blast search showed that RPS23RG1 shares some homology with the CRA_d isoform of human ATG10 [ATG10 autophagy related 10 homolog (S. cerevisiae). Gene ID 83734, identities = 33/40, 82%]. However, further analyses revealed that the ATG10 CRA_d isoform originated through a different mechanism from that of Rps23rg1–Rps23rg5 (see below).
Figure 1. Identification of the RPS23RG family. (A) Sequence alignment of the five RPS23RG family proteins. RPS23RG1–RPS23RG4 were found in mice (m). RPS23RG5 was found in chimpanzees (c). ‘.’, identical amino acids; ‘-’, (more ...)
To study whether Rps23rg family genes are expressed in mice, we carried out reverse transcription–PCR (RT–PCR) and found that Rps23rg1 was expressed in various mouse tissues (Fig. B). In addition, we found that Rps23rg2 was also expressed (Fig. B). However, we did not detect the expression of Rps23rg3 or Rps23rg4 in any tissue by RT–PCR with several pairs of primers (data not shown), implying that the two genes predicted by automatic computational analysis are either not real functional genes (i.e. they are processed pseudogenes) or they are expressed at very low levels that cannot be detected by RT–PCR. For Rps23rg5, we did not study whether it is expressed in chimpanzees due to limited resources. However, since Rps23rg5 was also predicted by automated computational analysis and we failed to detect its expression in humans by RT–PCR (data not shown), even though Rps23rg5 originated before the divergence between humans and chimpanzees (see below), it is possible that Rps23rg5 may also be a pseudogene without functional expression.
In our previous study, we found that the Rps23rg1
gene originated through retroposition of the mouse Rps23
). Therefore, we also compared gene sequences of other Rps23rg
members to those of Rps23
. Consistent with the results from the Rps23rg1
comparison, sequence alignments and analyses showed that Rps23rg2
gene sequences were highly homologous to the reverse and complementary sequences of mouse and chimpanzee Rps23
mRNA, respectively, and were all intronless within the homologous region (Fig. A and B). These results clearly demonstrate that all of the Rps23rg
gene family members originated through retroposition of Rps23
mRNA, but are reversely transcribed, relative to their parental genes.
Figure 2. Origination of the Rps23rg gene family through retroposition of Rps23 mRNA. The reverse and complementary (RC) sequences of mouse Rps23 (mRps23) and chimpanzee Rps23 (cRps23) cDNAs were aligned with mouse Rps23rg1–Rps23rg4 (A) and chimpanzee (more ...)
gene is highly conserved among species and belongs to the ribosomal protein family that is crucial for ribosome function (17
). It has been reported that human ribosomal protein genes have generated a large number of processed pseudogenes through retroposition (18
). Therefore, to further understand the origination of Rps23rg
family genes, we screened for all Rps23
-like sequences in human, mouse and rat genomes and constructed their phylogenic relationships. The results showed that retroposition of Rps23
occurred in all three species and occurred more frequently in rodents than in humans (Fig. ). The results also demonstrated that Rps23rg1
originated in mice after the divergence between mice and rats, whereas Rps23rg5
originated after the divergence between rodents and primates but before the divergence between humans and chimpanzees (Fig. ). Furthermore, to identify potential functional Rps23rg
homologs in humans, we selected fragments covering 100 Kb of the 5′ and the 3′ region adjacent to each of the identified Rps23
-like sequences in the human genome for gene prediction and found no Rps23rg
-like genes (data not shown). We also carried out RT–PCR with primers binding regions right next to these identified human Rps23
retroposition sites and failed to obtain any positive amplification (data not shown).
Figure 3. Phylogenic relationships of Rps23rg1–Rps23rg5, Rps23 mRNAs and other Rps23-like sequences in humans, rats and mice. Rps23-like sequences in human (h), rat (r) and mouse (m) genomes were identified by a blast search of GenBank with respective (more ...)
We focused our functional studies on Rps23rg1
since only these two genes were found to be expressed. RPS23RG2 is 24 amino acids shorter at the amino-terminus and 37 amino acids longer at the carboxyl-terminus than RPS23RG1. Otherwise, RPS23RG1 and RPS23RG2 have only one amino acid difference within the predicted transmembrane domain (Fig. A). Previously we showed that RPS23RG1 is a type Ib transmembrane protein and can be delivered to the cell surface (16
). Here we found that RPS23RG2 and its truncated form lacking the 37 amino acids at the carboxyl-terminus (RPS23RG2CΔ37) can also be delivered to the cell surface (Fig. B), implying that these RPS23RG family members have same subcellular localizations. Since overexpressed RPS23RG1 has been found to interact with adenylate cyclases and upregulate the cAMP level, activating PKA activity and thereby inhibiting GSK-3 activity, tau phosphorylation and Aβ generation (16
), we investigated whether RPS23RG2 has a similar function. We found that overexpressed RPS23RG2 also interacted with overexpressed adenylate cyclase 8 (Fig. A). In addition, overexpression of RPS23RG2 in human HeLa cells stably expressing human APP Swedish mutation upregulated PKA activity (with more CREB phosphorylation), decreased GSK-3α/β activities (with more GSK-3α/β phosphorylation), Aβ level and tau phosphorylation, and increased sAPPα release and APP carboxyl-terminal fragments (CTFs) levels (Fig. B). However, the effects of RPS23RG2 were much weaker than those of RPS23RG1 (Fig. B). In contrast, the truncated RPS23RG2CΔ37 form not only interacted with adenylate cyclase 8 (Fig. A), but also had effects on modulating PKA and GSK-3 phosphorylation/activity, APP processing/Aβ level and tau phosphorylation that were much stronger than those of RPS23RG2 and comparable to those of RPS23RG1 (Fig. B). These results imply that the extra carboxyl-terminus of RPS23RG2 may inhibit its effects on downstream signaling. On the other hand, the amino-terminal region of RPS23RG1 may not be functionally important since RPS23RG2CΔ37 lacks this region and has comparable effects to those of RPS23RG1.
Figure 4. RPS23RG family proteins interact with adenylate cyclases, activate PKA and reduce GSK-3 activity, Aβ level and tau phosphorylation. (A) RPS23RG family proteins interact with adenylate cyclase 8 (AC8). N2a cells were transfected with RPS23RG1, (more ...)
Since RPS23RG1 is a type Ib transmembrane protein and interacts with transmembranous adenylate cyclases (16
), we speculated that the transmembrane domain of the RPS23RG1 protein is important for its interaction with adenylate cyclases and for its activity. Therefore, we substituted the transmembrane domain of RPS23RG1 with that of APP or nicastrin, both of which are type I transmembrane proteins. As expected, co-immunoprecipitation studies showed that there is no interaction between adenylate cyclase 8 and the RPS23RG1 with an APP transmembrane domain substitution (Fig. A) or a nicastrin transmembrane domain substitution (data not shown). In addition, substitution of the RPS23RG1 transmembrane domain with that of APP or nicastrin abolished the effects of RPS23RG1 on upregulating phopshorylation of CREB and GSK-3α/β and reducing the level of Aβ (Fig. B).
Figure 5. The transmembrane domain is crucial for RPS23RG1's function. (A) The transmembrane domain is required for its interaction with AC. N2a cells were transfected with Myc-tagged RPS23RG1 or Myc-tagged RPS23RG1 carrying the APP transmembrane domain, together (more ...)
Blast of the GenBank database with the RPS23RG1 protein sequence revealed that the amino-terminus of RPS23RG1 shares homology with the carboxyl-terminus of the rare isoform CRA_d of the human ATG10 protein, whereas RPS23RG1 does not share any homology with the common ATG10 protein (Fig. A). However, detailed sequence analyses revealed that the human ATG10
CRA_d isoform did not originate through retroposition of the human Rps23
mRNA but rather acquired the homology by ‘hijacking’ an exonal domain of the human Rps23 gene: the human ATG10
gene is localized on chromosome 5 next to, but reversely transcribed relative to, the human RPS23
gene (Fig. A). Therefore, a part of exon 4 of human RPS23
is recruited by the ATG10
CRA_d isoform during its transcription and translation, encoding the ATG10 CRA_d isoform's carboxyl-terminus. Since Rps23
is highly conserved among different species and mouse Rps23rg1
is reversely transcribed relative to mouse Rps23
, the amino-terminus of RPS23RG1 becomes homologous to the carboxyl-terminus of ATG10 CRA_d isoform (Fig. A). The differences in origination suggest that the ATG10
CRA_d isoform should not be considered a member of the Rps23rg
family. Although ATG10 is thought to be an E2-like enzyme and involved in two ubiquitin-like modifications essential for autophagosome formation (19
), the exact function of ATG10 CRA_d isoform remains unknown. However, it is unlikely that ATG10 CRA_d isoform functions similarly to RPS23RG1 through the homologous domain, since the amino-terminus of RPS23RG1 is dispensable for its function (Fig. ). Indeed, overexpression of ATG10 CRA_d isoform, as well as ATG10, had no effects on promoting phosphorylation of CREB and GSK-3α/β or on reducing the level of Aβ as RPS23RG1 did (Fig. B).
Figure 6. ATG10 CRA_d isoform originated differently from RPS23RG family members and does not have the same effect. (A) The origin of the ATG10 CRA_d isoform is different from that of Rps23rg family members. The human ATG10 gene (gray boxes, CDS, white boxes, UTRs; (more ...)