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Identification of a Giardia Krr1 Homolog Gene and the Secondarily Anucleolate Condition of Giaridia Lamblia

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Identification of a Giardia Krr1 Homolog Gene and the Secondarily Anucleolate Condition of Giaridia Lamblia Identification of a Giardia 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 eukaryotes and to lie close to the transition between prokaryotes and eukaryotes, but several supporting features, such as lack of mitochondrion 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 eukaryote, 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 diplomonads 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).
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