Conservation and Developmental Control of Alternative Splicing in maebl Among Malaria Parasites Naresh Singh 1 , Peter Preiser 2 †, Laurent Re ´nia 3 †, Bharath Balu 1 John Barnwell 4 , Peter Blair 1 , William Jarra 2 , Tatiana Voza 5 , Ire ` ne Landau 5 and John H. Adams 1 * 1 Department of Biological Sciences, University of Notre Dame, 220 Galvin, PO Box 369 Notre Dame, IN 46556, USA 2 Division of Parasitology National Institute for Medical Research, The Ridgeway, Mill Hill NW7 1AA, UK 3 De ´partement d’Immunologie Institut Cochin, INSERM U567 CNRS 8104, Universite ´ Rene ´ Descartes, Ho ˆpital Cochin Ba ˆtiment Gustave Roussy 75014 Paris, France 4 Division of Parasitic Diseases National Center for Infectious Diseases, Centers for Disease Control & Prevention, Mailstop F-13, Bldg. 22B, 4770 Buford Highway, NE, Chamblee, GA 30341, USA 5 Museum National d’Histoire Naturelle, F-75231 Paris France Genes of malaria parasites and other unicellular organisms have larger exons with fewer and smaller introns than metaozoans. Such differences in gene structure are perceived to extend to simpler mechanisms for transcriptional control and mRNA processing. Instead, we discovered a surprisingly complex level of post-transcriptional mRNA processing in analysis of maebl transcripts in several Plasmodium species. Mechanisms for internal alternative cis-splicing and exon skipping were active in multiple life cycle stages to change exon structure in the deduced coding sequence (CDS). The major alternatively spliced transcript utilized a less favorable acceptor splice site, which shifted codon triplet usage to a different CDS with a hydrophilic C terminus, changing the canonical type I membrane MAEBL product to a predicted soluble isoform. We found that develop- mental control of the alternative splicing pattern was distinct from the canonical splicing pattern. Western blot analysis indicated that MAEBL expression was better correlated with the appearance of the canonical ORF1 transcript. Together these data reveal that RNA metabolism in unicellular eukaryotes like Plasmodium is more sophisticated than believed and may have a significant role regulating gene expression in Plasmodium. q 2004 Elsevier Ltd. All rights reserved. Keywords: malaria; Plasmodium; alternative splicing; MAEBL; ligand *Corresponding author Introduction Malaria is one of the most serious human diseases causing several million deaths and clinical illness in hundreds of millions of people every year. The biological complexity of these pathogenic proto- zoans is extraordinary in the intricate life cycles, as the malaria parasite must develop in two very different host organisms, infecting different cell types, and its motile stages must often traverse different tissues before infecting a new cell. 1 Never- theless, the Plasmodium genome is estimated to have less than 6000 genes and the number of unique genes is still less since a considerable part of the Plasmodium genome is occupied by multi-gene families (e.g. var , rif, stevor). 2,3 Compared to the 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. † P.P. and L.R. contributed equally to this work. Present addresses:P. Preiser, Nanyang Technological University, School of Biological Sciences, 60 Nanyang Drive, Singapore 637551; P. Blair, Department of Biology, Earlham College, 801 National Road West, Richmond, IN 47374, USA. Abbreviations used: AMA-1, apical membrane antigen- 1; DBP, Duffy antigen binding protein; DBL, duffy binding-like; EBA-175, erythrocyte binding antigen-175; ebl, erythrocyte binding-like; EBP, erythrocyte binding protein; IFA, indirect immunofluorescence assay; ORF, open reading frame. E-mail address of the corresponding author: jadams3@nd.edu doi:10.1016/j.jmb.2004.08.047 J. Mol. Biol. (2004) xx, 1–11 ARTICLE IN PRESS