Abstract
Giardia duodenalis is a parasite that colonises the intestines of humans and other vertebrates, causing diarrhoea and poor nutrient absorption. G. duodenalis is sometimes considered an early diverging eukaryote, and its genome exhibits simplified molecular machinery for many cellular processes, which makes it an interesting model to study. The spliceosome, one of the most complex molecular machines in the eukaryotic cell, is responsible for intron excision and exon splicing. Just over a decade ago, it was believed that the G. duodenalis genome did not contain introns or undergo splicing. Research now shows that this speculation was incorrect and that uncommon mechanisms, such as trans-splicing from different genes, occur. In silico studies of the parasite suggest the possibility of a simplified spliceosome and spliceosomal small nuclear RNA (snRNA) candidates; however, none of these components have been identified in vivo. Here, we developed a strategy to study the in vivo expression, interactions and localisation of these spliceosome components in G. duodenalis. Haemagglutinin (HA)-tagged SmB and SmD3 proteins, which form part of the spliceosome core, were overexpressed in the parasite. Immunoprecipitation with anti-HA revealed that the SmD3 protein is associated with the proteins SmB, SmD1, SmD2, SmE and SmF in vivo. In addition, the U1, U2 and U4 snRNA candidates reported previously were found in the protein complex, suggesting that these molecules are spliceosomal snRNAs of G. duodenalis and they contained a 2,2,7-trimethylguanosine modification at their 5′ end. Our data indicate that the actively expressed spliceosome in G. duodenalis is similar to that of highly evolved protists and higher animals.
Similar content being viewed by others
References
Alvarado ME, Wasserman M (2012) Calmodulin expression during Giardia intestinalis differentiation and identification of calmodulin-binding proteins during the trophozoite stage. Parasitol Res 110:1371–1380. doi:10.1007/s00436-011-2637-4
Best AA (2004) Evolution of eukaryotic transcription: insights from the genome of Giardia lamblia. Genome Res 14:1537–1547. doi:10.1101/gr.2256604
Chen XS, White WTJ, Collins LJ, Penny D (2008) Computational identification of four spliceosomal snRNAs from the deep-branching eukaryote Giardia intestinalis. PLoS One 3:e3106. doi:10.1371/journal.pone.0003106
Collins L, Penny D (2005) Complex spliceosomal organization ancestral to extant eukaryotes. Mol Biol Evol 22:1053–1066. doi:10.1093/molbev/msi091
Collins LJ, Macke TJ, Penny D (2004) Searching for ncRNAs in eukaryotic genomes : maximizing biological input with RNAmotif. J Integr Bioinform 1:6. doi:10.2390/biecoll-jib-2004-6
Dorn R, Reuter G, Loewendorf A (2001) Transgene analysis proves mRNA trans-splicing at the complex mod(mdg4) locus in Drosophila. Proc Natl Acad Sci U S A 98:9724–9729. doi:10.1073/pnas.151268698
Fischer SEJ, Bulter MD, Pan Q, Ruvkun G (2008) Trans-splicing in C. elegans generates the negative RNAi regulator ERI-6/7. Nature 455:491–496. doi:10.1038/nature07274.Trans
Frantz C, Ebel C, Paulus F, Imbault P (2000) Characterization of trans-splicing in euglenoids. Curr Genet 37:349–355. doi:10.1007/s002940000116
Franze de Fernandez MT, Hayward W, August JT (1972) Bacterial proteins required for replication of phage Qβ ribonucleic acid: purification and properties of host factor I, a ribonucleic acid-binding protein. J Biol Chem 247:824–831
Franzén O, Jerlström-Hultqvist J, Einarsson E, Ankarklev J, Ferella M, Andersson B, Svärd S (2013) Transcriptome profiling of Giardia intestinalis using strand-specific RNA-Seq. PLoS Comput Biol 9:e1003000. doi:10.1371/journal.pcbi.1003000
Gao X, Zhao X, Zhu Y, He J, Shao J, Su C, Zhang Y, Zhang W, Saarikettu J, Silvennoinen O, Yao Z, Yang J (2012) Tudor staphylococcal nuclease (Tudor-SN) participates in small ribonucleoprotein (snRNP) assembly via interacting with symmetrically dimethylated Sm proteins. J Biol Chem 287:18130–18141. doi:10.1074/jbc.M111.311852
Gómez V, Wasserman M (2016) Inhibición parcial de dos genes que codifican para proteínas spliceosomales en Giardia intestinalis. Biomédica 36:128–136. doi:10.7705/biomedica.v36i0.3068
Hossain M, Sharma S, Korde R, Kanodia S, Chugh M, Rawat K (2013) Organization of Plasmodium falciparum spliceosomal core complex and role of arginine methylation in its assembly. Malar J 12:1. doi:10.1186/1475-2875-12-333
Hudson AJ, Moore AN, Elniski D, Joseph J, Yee J, Russell A (2012) Evolutionarily divergent spliceosomal snRNAs and a conserved non-coding RNA processing motif in Giardia lamblia. Nucleic Acids Res 40:10995–11008. doi:10.1093/nar/gks887
Hudson AJ, Stark MR, Fast NM, Russell A, Rader S (2015) Splicing diversity revealed by reduced spliceosomes in C. merolae and other organisms. RNA Biol 12:1–8. doi:10.1080/15476286.2015.1094602
Jiménez-García LF, Zavala G, Chavez-Munguia B, Ramos-Godinez Mdel P, Lopez-Velazquez G, Segura-Valdez Mde L, Montanez C, Hehl AB, Arguello-Garcia R, Ortega-Pierres G (2008) Identification of nucleoli in the early branching protist Giardia duodenalis. Int J Parasitol 38:1297–1304. doi:10.1016/j.ijpara.2008.04.012
Kambach C, Walke S, Young R, Avis J, de la Fortelle E, Raker V, Lührmann R, Li J, Nagai K (1999) Crystal Structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. Cell 96:375–387. doi:10.1016/S0092-8674(00)80550-4
Kamikawa R, Inagaki Y, Hashimoto T (2014) Secondary loss of a cis-spliced intron during the divergence of Giardia intestinalis assemblages. BMC Res Notes 7:413. doi:10.1186/1756-0500-7-413
Kamikawa R, Inagaki Y, Tokoro M, Roger A, Hashimoto T (2011) Split introns in the genome of Giardia intestinalis are excised by spliceosome-mediated trans-splicing. Curr Biol 21:311–315. doi:10.1016/j.cub.2011.01.025
Keister DB (1983) Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg 77:487–488. doi:10.1016/0035-9203(83)90120-7
Korneta I, Magnus M, Bujnicki JM (2012) Structural bioinformatics of the human spliceosomal proteome. Nucleic Acids Res 40:7046–7065. doi:10.1093/nar/gks347
Komonyi O, Papai G, Enunlu I, Muratoglu S, Pankotai T, Kopitova D, Maroy P, Udvardy A, Boros I (2005) DTL, the Drosophila homolog of PIMT/Tgs1 nuclear receptor coactivator-interacting protein/RNA methyltransferase, has an essential role in development. J Biol Chem 280:12397–12404. doi:10.1074/jbc.M409251200
Li L, Wang CC (2005) Identification in the ancient protist Giardia lamblia of two eukaryotic translation initiation factor 4E homologues with distinctive functions. Eukaryot Cell 4:948–959. doi:10.1128/EC.4.5.948
Lloyd D, Harris JC (2002) Giardia: highly evolved parasite or early branching eukaryote? Trends Microbiol 10:122–127. doi:10.1016/S0966-842X(02)02306-5
Matera AG, Wang Z (2014) A day in the life of the spliceosome. Nat Rev Mol Cell Biol 15:108–121. doi:10.1038/nrm3742
Morrison HG, McArthur AG, Gillin FD, Aley SB, Adam RD, Olsen GJ, Best AA, Cande WZ, Chen F, Cipriano MJ, Davids BJ, Dawson SC, Elmendorf HG, Hehl AB, Holder ME, Huse SM, Kim UU, Lasek-Nesselquist E, Manning G, Nigam A, Nixon JE, Palm D, Passamaneck NE, Prabhu A, Reich CI, Reiner DS, Samuelson J, Svard SG, Sogin ML (2007) Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science (80-) 317:1921–1926. doi:10.1126/science.1143837
Mouaikel J, Verheggen C, Bertrand E, Tazi J, Bordonne R (2002) Hypermethylation of the cap structure of both yeast snRNAs and snoRNAs requires a conserved methyltransferase that is localized to the nucleolus. Mol Cell 9:891–901. doi:10.1016/S1097-2765(02)00484-7
Nageshan RK, Roy N, Hehl AB, Tatu U (2011) Post-transcriptional repair of a split heat shock protein 90 gene by mRNA trans-splicing. J Biol Chem 286:7116–7122. doi:10.1074/jbc.C110.208389
Niño CA, Prucca CG, Chaparro J, Luján HD, Wasserman M (2012) The ubiquitin-activating enzyme (E1) of the early-branching eukaryote Giardia intestinalis shows unusual proteolytic modifications and play important roles during encystation. Acta Trop 123:39–46. doi:10.1016/j.actatropica.2012.03.012
Niu XH, Hartshorne T, He XY, Agabian N (1994) Characterization of putative small nuclear RNAs from Giardia lamblia. Mol Biochem Parasitol 66:49–57. doi:10.1016/0166-6851(94)90035-3
Nixon JEJ, Wang A, Morrison HG, McArthur AG, Sogin ML, Loftus BJ, Samuelson J (2002) A spliceosomal intron in Giardia lamblia. Proc Natl Acad Sci U S A 99:3701–3705. doi:10.1073/pnas.042700299
Robertson HM, Navik JA, Walden KKO, Honegger HW (2007) The bursicon gene in mosquitoes: an unusual example of mRNA trans-splicing. Genetics 176:1351–1353. doi:10.1534/genetics.107.070938
Roy SW, Hudson AJ, Joseph J, Yee J, Russell AG (2012) Numerous fragmented spliceosomal introns, AT-AC splicing, and an unusual dynein gene expression pathway in Giardia lamblia. Mol Biol Evol 29:43–49. doi:10.1093/molbev/msr063
Russell AG, Shutt TE, Watkins RF, Gray MW (2005) An ancient spliceosomal intron in the ribosomal protein L7a gene (Rpl7a) of Giardia lamblia. BMC Evol Biol 5:45. doi:10.1186/1471-2148-5-45
Simoes-Barbosa A, Louly C, Franco OL, Rubio MA, Alfonzo JD, Johnson PJ (2008) The divergent eukaryote Trichomonas vaginalis has an m7G cap methyltransferase capable of a single N2 methylation. Nucleic Acids Res 36:6848–6858. doi:10.1093/nar/gkn706
Sogin M, Gunderson J, Elwood H, Alonso R, Peattie D (1989) Phylogenetic meaning of the kingdom concept: an unusual ribosomal RNA from Giardia lamblia. Science (80-) 243:75–77. doi:10.1126/science.2911720
Sutton RE, Boothroyd JC (1986) Evidence for trans splicing in trypanosomes. Cell 47:527–535, doi:10.1016/0092-8674(86)90617-3
Tian XF, Yang ZH, Shen H, Adam RD, Lu SQ (2010) Identification of the nucleoli of Giardia lamblia with TEM and CFM. Parasitol Res 106:789–793. doi:10.1007/s00436-009-1715-3
Yee J, Nash TE (1995) Transient transfection and expression of firefly luciferase in Giardia lamblia. Proc Natl Acad Sci U S A 92:5615–5619. doi:10.1073/pnas.92.12.5615
Zhong W, Ganem D (1997) Characterization of ribonucleoprotein complexes containing an abundant polyadenylated nuclear RNA encoded by Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8). J Virol 71:1207–1212
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Gómez, V., Wasserman, M. Interactions between Giardia duodenalis Sm proteins and their association with spliceosomal snRNAs. Parasitol Res 116, 617–626 (2017). https://doi.org/10.1007/s00436-016-5326-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-016-5326-5