**4.3 Additional paralogs of termination factors in several species**

Additional duplication of genes encoding termination factors have been found in several species (Figure 3). For example, an additional copy of eRF1 is present in some lineages of ciliates (Liang et al., 2001; Atkinson et al., 2008). These organisms differ from most eukaryotes by their reassignment of one or two stop codons to encode amino acids (Lozupone et al., 2001). UGA, for instance, encodes cysteine in *Euplotes* (Meyer et al., 1991). The presence of two copies of eRF1 in *Euplotes octocarinatus* may be associated with a different codon specificity of eRF1 proteins for UAA and UAG codons (Liang et al., 2001). Later studies showed that both eRF1a and eRF1b recognized UAA and UAG as stop codons

and provides prionogenic properties to the protein (Kushnirov & Ter Avanesyan, 1998). The same amino acid composition is also detected in the N-terminal domains of eRF3 in the kinetoplastid protists *L. major* and *Trypanosoma cruzi*, but this similarity is unlikely to be homologous (Atkinson et al., 2008). For termination of translation and maintenance of viability, only the C-terminal domain of eRF3 (homologous to elongation factor eEF1A) is necessary. eRF3 may have arisen in the early stages of eukaryotic evolution, since neither bacterial nor archaeal genomes contain homologues of eRF3 (Inagaki & Doolittle, 2000). Recent studies have shown that the functions of eRF3 can be performed in archaea by

The termination factor eRF3, preserving the functions typical of elongation factors (GTP-ase activity and interaction with the A-site of the ribosome), lost the capacity to bind tRNA but acquired the capacity to interact with eRF1 (Table 1). From this standpoint, elongation factor EF1A of archaea is functionally intermediate between elongation and termination factors: it acquired the ability to stimulate aRF1 while maintaining all the properties of an elongation factor (Saito et al., 2010). Termination factor eRF1 is a striking example of neofunctionalization, because it has acquired a variety of functions absent in elongation factors, including the ability to decode stop signals and to catalyze the release of nascent

EF-Tu RF1, RF2

eEF1A eRF1

Table 1. Functional homology between elongation and termination factors in Archaea,

Additional duplication of genes encoding termination factors have been found in several species (Figure 3). For example, an additional copy of eRF1 is present in some lineages of ciliates (Liang et al., 2001; Atkinson et al., 2008). These organisms differ from most eukaryotes by their reassignment of one or two stop codons to encode amino acids (Lozupone et al., 2001). UGA, for instance, encodes cysteine in *Euplotes* (Meyer et al., 1991). The presence of two copies of eRF1 in *Euplotes octocarinatus* may be associated with a different codon specificity of eRF1 proteins for UAA and UAG codons (Liang et al., 2001). Later studies showed that both eRF1a and eRF1b recognized UAA and UAG as stop codons

**Recognition of stop-signal in A-site of ribosome** 

**Function of acessory protein** 

> aEF1A (for aRF1), aEF1B (for aEF1A)

RF3 (for RF1 or RF2), EF-Ts (for EF-Tu)

eRF3 (for eRF1), eEF1B (for eEF1A)

peptides from eukaryotic ribosomes in response to stop codons.

**Archaea** aEF1A aEF1A aRF1

**4.3 Additional paralogs of termination factors in several species** 

**tRNA binding and delivering to the A-site of ribosome** 

**GTPbinding** 

> EF-Tu, RF3, EF-G

eEF1A, eRF3, eEF-2

aEF1A (Saito et al., 2010).

**Eubacteria** 

**Eucarya** 

Bacteria and Eukaryota

(Wang et al., 2010). The precise functions of each protein thus remain to be discovered. The plant *A. thaliana* has three paralogs of eRF1, all of which are able to rescue the *sup45-2(ts)*  mutation in *SUP45* (encoding eRF1) in *S. cerevisiae* (Chapman & Brown, 2004).

Another example of duplication, found only in some taxonomic groups, is the presence of two paralogous genes encoding eRF3 in mammals. In mammals, proteins homologous to eRF3 can be divided into two subfamilies based on the sequence of their N-termini. The first subfamily includes human hGSPT1 (or eRF3a) and mouse mGSPT1 (Hoshino et al., 1989; Hoshino et al., 1998; Jean-Jean et al., 1996), while the second subfamily includes human hGSPT2 (eRF3b) and mouse mGSPT2 (Hoshino et al., 1998; Jakobsen et al., 2001). Complementation experiments have shown that only *mGSPT2* is able to complement the *SUP35* gene (encoding eRF3) mutation (Le Goff et al., 2002). *GSPT2* is a paralog of *GSPT1* that has perhaps arisen as a result of retrotransposition of the *GSPT1* transcript into the genome of the common ancestor of mouse and human. *GSPT2* may thus be a functional retrogene (Zhouravleva et al., 2006). Both eRF3a and eRF3b are able to serve as termination factors in mammalian cells and interact with eRF1 (Chauvin et al., 2005). However, eRF3a is considered the main factor (Chauvin et al., 2005) that is expressed in all tissues, while eRF3b is detected only in the brain (Hoshino et al., 1998; Chauvin et al., 2005). This duplication event may not have led to the emergence of a new gene function but may have contributed to the complexity of regulatory processes by tissue-specific expression of these genes.

### **4.4 Subneofunctionalization in a family of termination factors gave rise to proteins participating in mRNA quality control**

A necessary condition of protein synthesis is to obtain functionally active proteins, so the control of accuracy of protein synthesis occurs at each stage of translation (Valente & Kinzy, 2003). The accuracy of initiation is achieved by proper identification of the start codon by a multifactorial initiation complex (Asano et al., 2001). Elongation requires the control of various events, including maintenance of the correct reading frame. Shifts in the reading frame occur at a frequency near 3 x 10-5 (Atkins et al., 1991) and may lead to the synthesis of non-functional products because shifts in the reading frame will often create a premature termination codon (PTC).

Eukaryotic cells possess a mechanism known as nonsense-mediated mRNA decay (**NMD**) that recognizes and degrades mRNA molecules containing premature termination codons (Amrani et al., 2006) (Figure 4). NMD is mediated by the trans-acting factors Upf1, Upf2 and Upf3, all of which directly interact with eRF3; only Upf1 interacts with eRF1 (Czaplinski et al., 1998; Wang et al., 2001). In addition to NMD, eukaryotic cells contain two additional mechanisms of mRNA quality control. No-go decay (**NGD**) releases ribosomes that are stalled on the mRNA (Doma & Parker, 2006). In yeast, NGD involves the proteins Hbs1 and Dom34 (Pelota in mammals). Another mechanism, non-stop decay (**NSD**), leads to the release of ribosomes that have read through the stop codon instead of terminating (Vasudevan et al., 2002). NSD has only been found in *S. cerevisiae* and involves the Ski7 protein (van Hoof et al., 2002). A common feature of these processes is that all involve the termination factors eRF1 and eRF3 (NMD) or their paralogs (Dom34/eRF1 and Hbs1/eRF3 in NGD; Ski7/eRF3 in NSD).

Hbs1 is a paralog of eEF1A and eRF3 (Wallrapp et al., 1998; Inagaki & Doolittle, 2000), while Dom34 is a paralog of eRF1 (Koonin et al., 1994; Davis & Engebrecht, 1998) (Figure 3). The C-terminus of Hbs1, homologous to that of eRF3, is sufficient to interact with Dom34, which assumes the same structure of the complex of two pairs of proteins (Hbs1-Dom34 and eRF3-

Gene Duplication and the Origin of Translation Factors 163

recognition of the mRNA stem (Graille et al., 2008). Lack of the Hbs1 protein in archaea is apparently compensated by its homolog aEF1A (Kobayashi et al,. 2010), which also performs the functions of eRF3 in archaeal termination of translation (Saito et al., 2010). In one more pathway of mRNA degradation, non-stop decay (NSD), participates In one more pathway of mRNA degradation, non-stop decay (NSD), participates Ski7 protein that is paralog of Hbs1 and eRF3 (Benard et al., 1999). This mechanism is necessary to destroy mRNAs lacking all termination codons (Frischmeyer et al., 2002; van Hoof et al., 2002). Ski7 protein that is paralog of Hbs1 and eRF3 (Benard et al., 1999). This mechanism is necessary to destroy mRNAs lacking all termination codons (Frischmeyer et al., 2002; van Hoof et al., 2002) Ski7, involved in NSD, arose from duplication of Hbs1 by WGD (Kellis et al., 2004) or by an independent duplication of Hbs1 before WGD and the subsequent loss in several species (Atkinson et al., 2008) (Figure 3). An interesting hypothesis links the appearance of Ski7 with the existence of the prion [*PSI+*] (Atkinson et al., 2008). [*PSI+*] is the aggregated (prion) form of the yeast protein Sup35 (eRF3) (Kushnirov & Ter Avanesyan, 1998). Formation of [*PSI+*] decreases the amount of functional Sup35, leading to the efficient read-through of nonsense mutations in ORFs (and possibly at the normal terminator codons) (Serio & Lindquist, 1999). The emergence of Ski7 in such organisms would thus create an additional system of mRNA quality control. However, [*PSI+*] formation has not been detected in the natural, industrial and clinical isolates of *Saccharomyces*. In addition, the prionic properties of Sup35 are conserved in various species of *Saccharomyces* as well as in *Candida albicans* and *Pichia methanolica* (Inge-Vechtomov et al., 2003), species in which Ski7 has not been found

Successive duplications of genes encoding elongation factors for translation led to the emergence of several protein complexes with different properties. The eRF1-eRF3 complex terminates translation, and the Dom34-Hbs1 complex is involved in the quality control of mRNA. Both eRF1 and eRF3 interact not only with each other but also with additional proteins. Some of these interactions are possibly mutually exclusive, and some of the proteins interacting with eRF1/eRF3 can be components of the complex terminating translation. Possible candidates for involvement in termination are poly(A) binding protein (PABP) and Upf proteins (Upf1, Upf2 and Upf3). Interaction of eRF3 with PABP links termination of translation with initiation (Hoshino et al., 1999), while interaction with Upf involves eRF proteins in nonsense-mediated decay (Amrani et al., 2006). The genetic data, derived mostly from *S. cerevisiae*, strongly suggest that the functions of eRF1 and eRF3 are not restricted to termination of translation (Inge-Vechtomov et al., 2003). Further studies are needed to characterize other non-translational functions of both proteins, as was shown for

This work was supported by the Russian Foundation for Basic Research (10-04-00237) and the Program of the Presidium of the Russian Academy of Sciences, The Origin and the

(Atkinson et al., 2008).

eEF1A (Mateyak & Kinzy, 2010).

**6. Acknowledgments** 

Evolution of the Biosphere.

**5. Conclusion** 

eRF1) (Carr-Schmid et al., 2002). Indeed, Hbs1 forms a complex with Dom34 and GTP (Dom34-Hbs1-GTP), similar to that of eRF1-eRF3-GTP (Hauryliuk et al., 2006; Graille et al., 2008; Chen et al., 2010; Shoemaker et al., 2010; van den Elzen et al., 2010). The central event of NGD is mRNA cleavage, and Dom34 has the necessary RNase activity (Lee et al., 2007; Graille et al., 2008), although the proposed endonuclease activity of Dom34 is not required for mRNA cleavage in NGD (Passos et al., 2009). Dom34 of *S. cerevisiae* consists of three domains, two of which are homologous to the corresponding domains in eRF1, while the Nterminal domain of Dom34 is different from that of eRF1 and is probably necessary for the

Fig. 4. Neofunctionalization of termination factors in mRNAs quality control systems. Three systems described for *S. cerevisiae* are shown. NSD (Non-stop decay) is responsible for the degradation of transcripts lacking stop codons. NGD (No-go decay) removes mRNA secondary structures that prevent translation. NMD (Nonsense-mediated decay) destroys transcripts containing nonsense mutations. See text for details.

recognition of the mRNA stem (Graille et al., 2008). Lack of the Hbs1 protein in archaea is apparently compensated by its homolog aEF1A (Kobayashi et al,. 2010), which also performs the functions of eRF3 in archaeal termination of translation (Saito et al., 2010).

In one more pathway of mRNA degradation, non-stop decay (NSD), participates In one more pathway of mRNA degradation, non-stop decay (NSD), participates Ski7 protein that is paralog of Hbs1 and eRF3 (Benard et al., 1999). This mechanism is necessary to destroy mRNAs lacking all termination codons (Frischmeyer et al., 2002; van Hoof et al., 2002). Ski7 protein that is paralog of Hbs1 and eRF3 (Benard et al., 1999). This mechanism is necessary to destroy mRNAs lacking all termination codons (Frischmeyer et al., 2002; van Hoof et al., 2002) Ski7, involved in NSD, arose from duplication of Hbs1 by WGD (Kellis et al., 2004) or by an independent duplication of Hbs1 before WGD and the subsequent loss in several species (Atkinson et al., 2008) (Figure 3). An interesting hypothesis links the appearance of Ski7 with the existence of the prion [*PSI+*] (Atkinson et al., 2008). [*PSI+*] is the aggregated (prion) form of the yeast protein Sup35 (eRF3) (Kushnirov & Ter Avanesyan, 1998). Formation of [*PSI+*] decreases the amount of functional Sup35, leading to the efficient read-through of nonsense mutations in ORFs (and possibly at the normal terminator codons) (Serio & Lindquist, 1999). The emergence of Ski7 in such organisms would thus create an additional system of mRNA quality control. However, [*PSI+*] formation has not been detected in the natural, industrial and clinical isolates of *Saccharomyces*. In addition, the prionic properties of Sup35 are conserved in various species of *Saccharomyces* as well as in *Candida albicans* and *Pichia methanolica* (Inge-Vechtomov et al., 2003), species in which Ski7 has not been found (Atkinson et al., 2008).
