Molecular Parasitology-Anything and Everything about the Spliced Leader RNA

     Our research focuses on the nuclear gene expression of the kinetoplastid protozoa, commonly known as trypanosomes. These mainly parasitic protists are found in a diverse range of host organisms, including insects, fish, molluscs, plants, reptiles and mammals, and are the causative agents of medical and agricultural afflictions. In humans, diseases such as African Sleeping Sickness, Leishmaniasis, and Chagas Disease are caused by trypanosomatid infections, afflicting millions of people worldwide.

     Our work is specialized in the characterization, expression and action of a key player in the expression of all nuclear protein coding genes called the Spliced Leader (SL) RNA, or mini-exon RNA. The ~100-nt SL RNA contains a 39-nt exon that is transferred via a trans-splicing reaction to the 5' end of every nuclear mRNA. The trans-splicing of the SL exon is critical for trypanosomatids, as their mRNAs are encoded in a polycistronic fashion and cannot receive their 5' caps structures directly. Because this form of trans-splicing is not found in the human or insect hosts, a complete understanding of the genesis of the SL RNA and the trans-splicing process may lead to clinical treatments for trypanosome infections.

     The SL RNA gene is found in a head-to-tail tandem array, which we have used to develop a universal trypanosomatid PCR assay. Using this assay we have amplified over 50 different trypanosomatids and close relatives, providing us with a valuable database for inter-genus and inter-species comparisons, as well as a powerful diagnostic tool. Perhaps the most interesting finding is the extent to which the 39-nt SL sequence is conserved, implying a critical role for this sequence block that extends throughout the Family.

     The SL RNA gene represents the first and only RNA polymerase II transcript with a defined promoter in the trypanosomatids to date. We have identified two elements of the SL RNA gene promoter of lizard leishmania Leishmania tarentolae to the nucleotide level and are in the process of biochemically purifying interacting factors, as well as cloning likely transcription factors as they appear in the genome database. Our experiments have examined the SL RNA gene transcription termination element, along with several processing steps for the SL RNA itself including 3' trimming and 5' end methylations resulting in the 'cap4' structure. Our current model for the processing of the SL RNA includes a cytoplasmic maturation stage, a matter of some debate in our field.

      One of the questions we would like to resolve is the role of the conserved sequence element within the SL exon. Since we have demonstrated that it is not required for transcription, termination, 3'- or 5'-processing, or trans-splicing itself, we are exploring the role of the SL in translation and in nucleosome structure.

     Campbell, D.A., Thomas, S. and Sturm, N.R. (2003) Transcription in the kinetoplastid protozoa: why be normal?. Microbes and Infection In Press.

     Zeiner, G.M., Sturm, N.R. and Campbell, D.A. (2003) The Leishmania tarentolae spliced leader contains determinants for association with polysomes. The Journal of Biological Chemistry 278: In Press.

     Grisard, E.C., Sturm, N.R. and Campbell, D.A. (2003) A new species of trypanosomes isolated from South American bats, Trypanosoma desterris sp. n. Parasitology 127: 265-271.

     Zeiner, G.M., Sturm, N.R. and Campbell, D.A. (2003) Exportin 1 mediates nuclear export of the kinetoplastid Spliced Leader RNA. Eukaryotic Cell 2: 222-230.

     Sturm, N.R., Vargas, N.S., Westenberger, S.J., Zingales, B. and Campbell, D.A. (2003) Evidence for multiple hybrid groups in Trypanosoma cruzi. International Journal for Parasitology 33: 269-279.

     Santos, S.S., Cupolillo, E., Junquiera, A., Coura, J.R., Jansen, A., Sturm, N.R., Campbell, D.A. and Fernandes, O. (2002) Genetic diversity of Brazilian Trypanosoma cruzi isolates and phylogenetic positioning of zymodeme 3 based on the internal transcribed spacer of the ribosomal gene. Annals of Tropical Medicine and Parasitology 96: 755-764.

     Yu, M.C., Orlando, T.C., Sturm, N.R., Zhou, L., Saito, R.M., Floeter-Winter, L.M. and Campbell, D.A. (2002) Two distinct functional spliced leader RNA gene arrays in Leishmania tarentolae are found in several lizard Leishmania species. International Journal for Parasitology 32: 1411-1422.

     Santana, D.M., Borja-Cabrera, G.P., Paraguai de Souza, E., Sturm, N.R., Palatnik de Sousa, C.B., and Campbell, D.A. (2002) Nucleoside hydrolase from Leishmania (L.) donovani is an antigen diagnostic for visceral leishmaniasis. Molecular and Biochemical Parasitology 120: 315-319.

     Orlando, T.C., Rubio, M.A.T., Sturm, N.R., Campbell, D.A., and Floeter-Winter, L.M. (2002) Intergenic and external transcribed spacers of ribosomal RNA genes in lizard-infecting Leishmania: Molecular structure and phylogenetic relationship to mammal-infecting Leishmania in the subgenus Leishmania (Leishmania). Memórias do Instituto Oswaldo Cruz 97: In Press.

     Dollet, M., Sturm, N.R., Ahomadegbe, J.-C. and Campbell D.A. (2001) Kinetoplast DNA minicircles of phloem-restricted Phytomonas associated with wilt diseases of coconut and oil palms have a two-domain structure. FEMS Microbiology Letters 205: 65-69.

     Santana, D.M., Lukeš, J., Sturm, N.R. and Campbell, D.A. (2001) Two sequence classes of kinetoplastid 5S ribosomal RNA gene revealed among Bodonid spliced leader RNA gene arrays. FEMS Microbiology Letters 204: 233-237.

     Sturm, N.R., Maslov, D.A., Grisard, E.C. and Campbell, D.A. (2001) Diplonema spp. possess spliced leader RNA genes similar to the Kinetoplastida. Journal of Eukaryotic Microbiology 48: 325-331.

     Dollet, M., Sturm, N.R. and Campbell, D.A. (2001) The spliced leader RNA gene array in phloem-restricted plant trypanosomatids (Phytomonas) partitions into two major groupings: epidemiological implications. Parasitology 122: 289-297.

     Fernandes, O., Santos, S.S., Cupolillo, E., Mendonça, M.B.A., Junqueira, A., Santos, L.C., Derré, R., Sturm, N.R., Naiff, R.D., Barrett, T.V., Campbell, D.A. and Coura, J.R. (2001) A mini-exon multiplex polymerase chain reaction to distinguish the major groups of Trypanosoma cruzi and T. rangeli in the Brazilian Amazon. Transactions of the Royal Society of Hygiene and Tropical Medicine 95: 97-99.

     Yu, M.C., Roberts, T.G., Sturm, N.R. and Campbell, D.A. (2000) In vitro transcription of mutated Leishmania tarentolae spliced leader RNA genes approximates in vivo patterns. Molecular and Biochemical Parasitology 111: 391-399.

     Dollet, M., Sturm, N.R., Sánchez-Moreno, M. and Campbell, D.A. (2000) 5S ribosomal RNA gene sequences define at least eight groups of plant trypanosomatids (Phytomonas spp.): phloem-restricted pathogens form a distinct section. Journal of Eukaryotic Microbiology 47: 569-574.

     Pacheco, R.S., Fernandes, O., Salina, G., Segura, I., Momen, H., Degrave, W., Saravia, N.G. and Campbell, D.A. (2000) Intraspecific heterogeneity in the mini-exon gene localization of Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis from Colombia. Journal of Parasitology 86: 1250-1253.

     Grisard, E.C., Steindel, M., Shaw, J.J., Ishikawa, E.A.Y., Carvalho Pinto, C.J., Eger-Mangrich, I., Toma, H.K., Lima, J.H., Romanha, A.J. and Campbell, D.A. (2000) Characterization of Leishmania sp. strains isolated from autochthonous cases of human cutaneous leishmaniasis in Santa Catarina State, southern Brazil. Acta Tropica 74: 89-93.

     Fernandes, O., Santos, S.S., Junqueira, A.C.V., Jansen, A.M., Cupolillo, E., Campbell, D.A., Zingales, B. and Coura, J.R. (1999) Populational heterogeneity of Brazilian Trypanosoma cruzi isolates revealed by the mini-exon and ribosomal spacers. Memórias do Instituto Oswaldo Cruz 94: 195-197.

     Sturm, N.R. and Campbell, D.A. (1999) The role of intron structures in trans-splicing and cap 4 formation for the Leishmania Spliced Leader RNA. The Journal of Biological Chemistry 274: 19361-19367.

     Grisard, E.C., Campbell, D.A. and Romanha, A.J. (1999) Mini-exon gene sequence polymorphism among Trypanosoma rangeli strains isolated from different geographical regions. Parasitology 118: 375-382.

     Sturm, N.R., Yu, M.C., and Campbell, D.A. (1999) Transcript termination and 3'-end processing of the Spliced Leader RNA in kinetoplastids. Molecular and Cellular Biology 19: 1595-1601.

     Fernandes, O., Mangia, R., Lisboa, C.V., Pinho, A.P., Morel, C.M., Zingales, B., Campbell, D.A. and Jansen, A.M. (1999) The complexity of the sylvatic cycle of Trypanosoma cruzi in Rio de Janeiro state (Brazil) revealed by the non-transcribed spacer of the mini-exon gene. Parasitology 118: 161-166.

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