title: Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing complexes.
abstract: The genome of the human intestinal parasite Entamoeba histolytica contains nearly 3000 introns and bioinformatic predictions indicate that major and minor spliceosomes occur in Entamoeba. However, except for the U2-, U4-, U5- and U6 snRNAs, no other splicing factor has been cloned and characterized. Here, we HA-tagged cloned the snRNP component U1A and assessed its expression and nuclear localization. Because the snRNP-free U1A form interacts with polyadenylate-binding protein, HA-U1A immunoprecipitates could identify early and late splicing complexes. Avoiding Entamoeba's endonucleases and ensuring the precipitation of RNA-binding proteins, parasite cultures were UV cross-linked prior to nuclear fraction immunoprecipitations with HA antibodies, and precipitates were subjected to tandem mass spectrometry (MS/MS) analyses. To discriminate their nuclear roles (chromatin-, co-transcriptional-, splicing-related), MS/MS analyses were carried out with proteins eluted with MS2-GST-sepharose from nuclear extracts of an MS2 aptamer-tagged Rabx13 intron amoeba transformant. Thus, we probed thirty-six Entamoeba proteins corresponding to 32 cognate splicing-specific factors, including 13 DExH/D helicases required for all stages of splicing, and 12 different splicing-related helicases were identified also. Furthermore 50 additional proteins, possibly involved in co-transcriptional processes were identified, revealing the complexity of co-transcriptional splicing in Entamoeba. Some of these later factors were not previously found in splicing complex analyses.
  
  
  CONCLUSIONS
  Numerous facts about the splicing of the nearly 3000 introns of the Entamoeba genome have not been unraveled, particularly the splicing factors and their activities. Considering that many of such introns are located in metabolic genes, the knowledge of the splicing cues has the potential to be used to attack or control the parasite. We have found numerous new splicing-related factors which could have therapeutic benefit. We also detected all the DExH/A RNA helicases involved in splicing and splicing proofreading control. Still, Entamoeba is very inefficient in splicing fidelity, thus we may have found a possible model system to study these processes.
authors.affiliation: Departament of Biochemistry, CINVESTAV, México D.F., Mexico. Electronic address: jvaldes@cinvestav.mx.
authors.forename: Jesús
authors.initials: J
authors.lastname: Valdés
authors.affiliation: Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Nozaki
authors.affiliation: Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Emi
authors.initials: E
authors.lastname: Sato
authors.affiliation: University of Tsukuba, Graduate School of Life and Environmental Sciences, Tsukuba, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Chiba
authors.affiliation: Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Nakada-Tsukui
authors.affiliation: Department of Molecular Biomedicine, CINVESTAV, México D.F., Mexico.
authors.forename: Nicolás
authors.initials: N
authors.lastname: Villegas-Sepúlveda
authors.affiliation: Department of Biotechnology and Biochemistry, CINVESTAV Unidad Irapuato, Irapuato, Guanajuato, Mexico.
authors.forename: Robert
authors.initials: R
authors.lastname: Winkler
authors.affiliation: Posgrado en Ciencias Genómicas, UACM, México D.F., Mexico.
authors.forename: Elisa
authors.initials: E
authors.lastname: Azuara-Liceaga
authors.affiliation: Departament of Biochemistry, CINVESTAV, México D.F., Mexico.
authors.forename: María Saraí
authors.initials: MS
authors.lastname: Mendoza-Figueroa
authors.affiliation: University of Tsukuba, Graduate School of Life and Environmental Sciences, Tsukuba, Japan.
authors.forename: Natsuki
authors.initials: N
authors.lastname: Watanabe
authors.affiliation: Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan; University of Tsukuba, Graduate School of Life and Environmental Sciences, Tsukuba, Japan; Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City, Philippines.
authors.forename: Herbert J
authors.initials: HJ
authors.lastname: Santos
authors.affiliation: Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Saito-Nakano
authors.affiliation: Departament of Biochemistry, CINVESTAV, México D.F., Mexico.
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Galindo-Rosales
mesh_terms: D017398:Alternative Splicing; D002467:Cell Nucleus / Q000378:metabolism; D030562:Databases, Protein; D004748:Entamoeba histolytica / Q000378:metabolism*; D007438:Introns; D020543:Proteome; D040901:Proteomics; D020365:RNA Helicases / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome; D012322:RNA Precursors; D012333:RNA, Messenger; D017413:Ribonucleoprotein, U2 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D020365:RNA Helicases
keywords: DExH/A RNA helicases; Entamoeba histolytica; Splice sites; Splicing; Splicing factors; mRNP