Identification of a krr1 Homolog Gene and the Secondarily Anucleolate Condition of Giaridia lamblia

De-Dong Xin,* Jian-Fan Wen,* De He,* and Si-Qi Luà *Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China; Graduate School of the Chinese Academy of Sciences, Beijing, China; and àCapital University of Medical Sciences, Beijing, China

Giaridia lamblia was long considered to be one of the most primitive and to lie close to the transition between prokaryotes and eukaryotes, but several supporting features, such as lack of and Golgi, have been challenged recently. It was also reported previously that G. lamblia lacked nucleolus, which is the site of pre-rRNA processing and ribosomal assembling in the other eukaryotic cells. Here, we report the identification of the yeast homolog gene, krr1, in the anucleolate , G. lamblia. The krr1 gene, encoding one of the pre-rRNA processing proteins in yeast, is actively transcribed in G. lamblia. The deduced protein sequence of G. lamblia krr1 is highly similar to yeast KRR1p that contains a single-KH domain. Our database searches indicated that krr1 genes actually present in diverse Downloaded from https://academic.oup.com/mbe/article/22/3/391/1075989 by guest on 24 September 2021 eukaryotes and also seem to present in Archaea. However, only the eukaryotic homologs, including that of G. lamblia, have the single-KH domain, which contains the conserved motif KR(K)R. Fibrillarin, another important pre-rRNA processing protein has also been identified previously in G. lamblia. Moreover, our database search shows that nearly half of the other nucleolus-localized protein genes of eukaryotic cells also have their homologs in Giardia. Therefore, we suggest that a common mechanism of pre-RNA processing may operate in the anucleolate eukaryote G. lamblia and in the other eukaryotes and that like the case of ‘‘lack of mitochondrion,’’ ‘‘lack of nucleolus’’ may not be a primitive feature, but a secondarily evolutionary condition of the parasite.

Introduction In eukaryotes, ribosome biosynthesis, a process that contacts (Musco et al. 1996). KH domains occur in a wide entails rDNA transcription, pre-rRNA processing and variety of proteins, and they share the only property of rRNA assembly with ribosomal proteins, occurs in the associating with RNA. specialized subnuclear compartment, the nucleolus. The Giardia lamblia is one of the most widespread pre-rRNA processing is a complex process in which a large intestinal protozoan parasites. It has long been considered number of proteins and small nucleolar RNAs (snoRNAs) as one of the most primitive extant eukaryotes because of its are involved. These proteins include rRNA-modifying initially perceived lack of mitochondria and of some other enzymes, endonucleases and exonucleases, RNA helicases, membrane-bounded organelles typical of eukaryotic cells and components of small nucleolar ribonucleoprotein com- (Gillin, Reiner, and McCaffery 1996) and its early branching plexes (Kressler, Linder, and de La Cruz 1999). Among position in many molecular phylogenetic trees (Roger 1999; them, fibrillarin, nucleolin, and NOP52 have been well Adam 2001). It seems that this organism has diverged before characterized (see http://npd.hgu.mrc.ac.uk/compartments/ the acquisition of these cellular organelles and, thus, might nucleolus.html). provide insightful clues into the early evolution of The krr1 gene, which encodes KRR1p located in the eukaryotes. However, this opinion has been challenged by nucleolus, was first identified in yeast (Gromadka et al. several recent studies, such as the discoveries of mitochon- 1996; Gromadka and Rytka 2000). KRR1p serves as a pre- drial origin genes (e.g., cpn 60, mtHsp70, IscS, and IscU) rRNA processing machinery protein that contributes to the (Roger et al. 1998; Tachezy, Sanchez, and Muller 2001; process and synthesis of 18S and 25S rRNA (Gromadka Arisue et al. 2002; Tovar et al. 2003) and the mitochondrial and Rytka 2000; Sasaki, Toh, and Kikuchi 2000). A remnant organelle (mitosome) (Tovar et al. 2003). Further- KRR1p homolog, DBE, was later identified in Drosophila more, its basal position in phylogenetic trees was argued as and was revealed as an important protein for the pro- a result of a long-branch attraction (LBA) artifact, which cessing of both 18S and 28S rRNA (Chan, Brogna, and leads to their misidentification with distant prokaryotic O’Kane 2001). Homologous gene sequences were also outgroups (Philippe, Germot, and Moreira 2000). found in expressed sequence tags from several other In previous reports, it was also showed that eukaryotes: Caenorhabditis elegans, Oryza sativa, and G. lamblia lacked nucleoli, and no nucleolar skeleton Homo sapiens (Gromadka et al. 1996). All these KRR1p structure was found in its nucleus during investigation of homologs contain a putative K homology (KH) domain, the nuclear matrix.This was also regarded as one of the a 70 to 100 amino acid module that was originally iden- primitive features of the organism (Li, He, and Chen 1997; tified as a repeating sequence in heterogeneous nuclear Wen and Li 1998; Li 1999; Adam 2001). In practice, it is ribonucleoprotein K (Siomi et al. 1993). It is an RNA- also hard to find obvious nucleoli in other binding motif that is thought to make direct protein-RNA and their close relatives retortmonads, although there is suspicious denser material located against the nuclear en- velope in some EM pictures (Brugerolle 1973; Brugerolle, Key words: Giardia lamblia, krr1 gene, nucleolus, rRNA-processing, Joyon, and Oktem 1974; Silberman et al. 2002). However, evolution. ‘‘lacking nucleoli’’ in Giardia has been more studied and E-mail: [email protected]. emphasized by some authors as a primitive feature. Here, Mol. Biol. Evol. 22(3):391–394. 2005 doi:10.1093/molbev/msi052 we use the word ‘‘anucleolate,’’ which was first used by Advance Access publication November 17, 2004 Elsdale, Fischberg, and Smith (1958) to feature the mutant

Molecular Biology and Evolution vol. 22 no. 3 Ó Society for Molecular Biology and Evolution 2004; all rights reserved. 392 Xin et al.

Table 1 KRR1p Homologs Obtained by Searching GenBank Accession Species Number Taxon Abbreviation Homo sapiens NP_008974.4 Metazoa Hsap Rattus norvegicus XP_235128.1 Metazoa Rnor Mus musculus BAC27022.1 Metazoa Mmus Xenopus laevis AAH41273.1 Metazoa Xlae Drosophila AAF51440.1 Metazoa Dmel melanogaster Anopheles gambiae EAA12217.2 Metazoa Agam Caenorhabditis elegans NP_504837.1 Metazoa Cele Schistosoma japonicum AAP06389.1 Metazoa Sjap FIG.1.—PCR and RT-PCR demonstrate that the krr1 gene exists Saccharomyces NP_009872.1 Fungi Scer in Giardia genome and is actively transcribed. RT-PCR primers: down cerevisiae Downloaded from https://academic.oup.com/mbe/article/22/3/391/1075989 by guest on 24 September 2021 stream primer selectively matches the poly (A) tail of mRNA; upstream Schizosaccharomyces NP_596073.1 Fungi Spom primer matches the upstream of the krr1 ORF. PCR primers: upstream pombe primer was the same to the upstream primer of the RT-PCR; down- Neurospora crassa XP_327327.1 Fungi Ncra stream primer matches the tail of krr1 ORF. M indicates marker. Lane 1, Aspergillus nidulans EAA58384 Fungi Anid PCR using gDNA of G. lamblia as templates. Lane 2, RT-PCR using Magnaporthe grisea EAA52921 Fungi Mgri total RNA of G. lamblia as templates. Gibberella zeae EAA74559 Fungi Gzea Encephalitozoon NP_584734.1 Fungi Ecun of Xenopus that lacks typical nucleoli, to describe the cuniculi situation of Giardia. In this brief communication, we tried Oryza sativa AAP54059.1 Plants Osat Arabidopsis thaliana AAM64563.1 Plants Atha to determine whether the ‘‘anucleolate’’ situation is indeed Plasmodium falciparum NP_473002.1 Protists Pfal a primitive feature of the organism by investigating the Plasmodium yoelii EAA15212.1 Protists Pyoe krr1 homolog gene and its transcription in Giardia and Guillardia theta NP_113423.1 Protists Gthe comparing them with those of the other eukaryotes. Methanococcus NP_247417.1 Archaebacteria Mjan jannaschii Methanopyrus NP_613798.1 Archaebacteria Mkan Materials and Methods kandleri We first used yeast KRR1 as a query to Blast the G. lamblia genome database (www.mbl.edu/Giardia) and TKPYKPAKVAKRK) and 245 to 260 (KKNTKPYK- found an open reading frame (ORF) ranging from position PAKVAKRKR), indicating its nuclear localization. 114.922 to position 107.317 in the contig 735. Because Sequence searches using the Blast program and some features of the G. lamblia genome can lead to errors COGnitor showed that 22 krr1 homologs appeared in 22 in assembled contigs, we designed a pair of primers, which eukaryotes ranging from protists, fungi, and plants to corresponded to the upstream and downstream regions of metazoa, and two krrl homologs appear in two archaebacteria the ORF, respectively, to verify the ORF sequence through (Methanococcus jannaschii and Methanopyrus kandleri) PCR and sequencing. The segment we sequenced is (table 1). However, no krr1 homolog was found in eubacteria. identical to the identified ORF. To examine whether the Analysis by SMART program (http://smart.embl-heidelberg. gene is actively transcribed, we performed a RT-PCR de/) revealed that all the eukaryotic KRR1p homologs have (using freshly prepared total RNA from trophozoites) and a single KH domain, whereas the archaeal homologs both then cloned and sequenced the products (fig 1). The possess two KH domains: one adjacent to the N-terminal, the sequence we obtained has a poly (A) tail beginning at 37 nt other adjacent to the C-terminal. The latter is more similar to downstream of the stop codon (TAA) and a polyadenyla- that of eukaryotic KRR1p homologs except without the KRR tion signal AGTAAA typical to other reported Giardia motifs located in the a1 helix (fig. 2). genes. Thus, these data clearly demonstrated that Giardia Proteins with KH domains usually have more than one has a krr1 gene (GenBank accession number AF541964) such domain (between two and 14) (Musco et al. 1996). The homolog, and it is actively transcribed. only known exception is the STAR subfamily proteins, which have only one KH domain (Vernet and Artzt 1997). Results and Discussion We compared the STAR subfamily proteins with our euka- ryotic KRR1p homologs and found that sequence similarity Analysis of the deduced protein sequence showed between them was limited to the KH domain. Figure 2 41.62% identity and 70.96% similarity to yeast KRR1p. It shows KH domain alignment of five STAR proteins with has a basic isoelectric point (9.54) calculated by ProtParam our KRR1p homologs. In comparison with the KH domains tool (http://us.expasy.org/tools/protparam.html), which is in STAR protein, the KH domain in KRR1p has a big similar to that of yeast KRR1p (9.42). Similar to yeast deletion between the b2 and b3 sheets. Moreover, all eukary- KRR1p, the Giardia KRR1p contains a conserved KH otic KRR1p homologs have a unique highly conserved motif located at 135 aa to 207 aa, which is 73 aa in length KR(K)R motif, which is located in the a1 helix and carries with 52% identity to the yeast KRR1p KH domain. Scan- strong positive charges, whereas STAR proteins do not Prosite program (http://au.expasy.org/tools/scanprosite/) have such a motif. The unique single-KH domain of all analyses indicated the presence of two bipartial nuclear these KRR1p homologs suggests a novel subfamily in the localization signals (NLS) at regions 244 to 260 (KKKN- KH domain protein family. Giardia krr1 Gene and Secondarily Anucleolate Condition 393 Downloaded from https://academic.oup.com/mbe/article/22/3/391/1075989 by guest on 24 September 2021

FIG. 2.—KH domain alignment of STAR proteins with KRR1p homologs. In comparison with STAR protein KH domains, all KRR1p KH domains have a big deletion between the b2 and b3 sheets. Different from those of archaebacteria, all eukaryote KRR1p have a unique highly conserved KR(K)R motif (frame), which is located in the a1 helix and carries strong positive charges. The aligned KH domains in the SMART database were used to guide our alignment. The secondary structure is determined according to the crystal structure of 1KHM (PDB entry). b indicates beta sheet; a indicates alpha helix. The accession numbers of the five STAR proteins are as follows: Sam68, NP_006550; GRP33, P13230; Quaking-1, AAC52491.1; GLD-1, NP_492143; How, AAB51251.1. The accession numbers of KRR1p homologs and the abbreviations are in table 1.

Because KRR1p physically and functionally interacts processing and ribosomal assembly should be similar to with a protein Kri1p to form a complex, which is required those of the other eukaryotes and dissimilar to those of for 40S ribosome biogenesis in the nucleolus in yeast prokaryotes. Therefore, we suggest that like the initially (Sasaki, Toh, and Kikuchi 2000), we also used Krilp as perceived ‘‘lack of mitochondria,’’ the ‘‘lack of nucleolus’’ a query to Blast genomic data available in GenBank. We is not a primitive feature of G. lamblia but probably arose identified Kri1p homologs in both G. lamblia and other secondarily. Indeed, in addition to the discovery of mito- eukaryotic lineages but not in eubacteria or archaebacteria some (Tovar et al. 2003), accumulating evidence, such as (data not shown). the discoveries of intron and Golgi in Giardia (Lujan et al. Basing on the above analyses, we suggest that the 1995; Dacks and Doolittle 2001; Nixon et al. 2002), has anucleolate eukaryote, G. lamblia, has a homolog of yeast also proved some previously so-called ‘‘primitive fea- KRR1p, which is present in diverse eukaryotes, not in tures’’ to be shaky. Our present work also implies that the prokaryotes. anucleolate feature of other diplomonads and their close Fibrillarin, another important protein involved in pre- relatives might arise secondarily. To confirm rRNA processing and ribosome assembly, is conserved in this, it is necessary to find nucleolar homologs in them. both eukaryotes and archaebacteria (Tollervey et al. 1991, 1993). It had also been identified in G. lamblia previously Acknowledgments (Narcisi, Glover, and Fechheimer 1998). The identi- fied Giardia fibrillarin has a GAR domain (glycine and In this work, the database of Giardia lamblia Genome arginine–rich N-terminal domain), which is found only in Project (http://jbpc.mbl.edu/Giardia-HTML/), Marine Bio- eukaryotic fibrillarins and clusters with its eukaryotic homo- logical Laboratory at Woods Hole was used. We thank logs in phylogenetic trees (Narcisi, Glover, and Fechheimer authors of the database. This work was supported by grants 1998; Hickey, Macario, and Conway de Macario 2000). (30070362, 30170135, 30021004) from the National We next used 207 S. cerevisiae nucleolus-localized Natural Science Foundation of China to J.F.W and a grant proteins (http://mips.gsf.de/) as queries to Blast the G. lamblia (KSCX2-SW-101C) from the Chinese Academy of Sci- genomic database, and 100 potential homologs (E value ,1e- ences to J.F.W. 05 as cutoff) were identified. Most of them are similar to those involved in pre-rRNA processing and ribosomal assembly (see Literature Cited table 1 in Supplementary Material online). Adam, R. D. 2001. Biology of Giardia lamblia. Clin. Microbiol. Rev. 14:447–475. Conclusion Arisue, N., L. B. Sanchez, L. M. Weiss, M. Muller, and T. Hashimoto. 2002. Mitochondrial-type hsp70 genes of the Overall, our data imply that although G. lamblia amitochondriate protists, Giardia intestinalis, Entamoeba lacks nucleolar structure, the mechanism of its pre-rRNA histolytica and two microsporidians. Parasitol. Int. 51:9–16. 394 Xin et al.

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