Control mechanisms of tubulin gene expression in Trypanosoma cruzi.
alpha- and beta-Tubulin mRNAs are three to six-fold more abundant in the epimastigote forms than in trypomastigote and amastigote forms of Trypanosoma cruzi. It has been previously shown that the increased abundance of alpha- and beta-tubulin mRNAs found in epimastigotes is due to an increase in their half-lives. By analysing soluble and cytoskeletal protein fractions of the parasite, we found an inverse correlation between tubulin mRNA and the protein levels of free alpha- and beta-tubulin subunits, which are more abundant in trypomastigotes and amastigotes than in epimastigotes. Here we investigated a possible autoregulatory mechanism responsible for the differential accumulation of tubulin mRNAs in T. cruzi by treating epimastigotes with vinblastine and taxol, drugs that disrupt microtubule dynamics by different mechanisms: vinblastine causes significant depolymerisation of tubulin whereas taxol stabilises microtubules. Vinblastine treatment caused significant morphological alterations in epimastigotes whereas taxol does not alter the parasite morphology. Vinblastine, but not taxol, had a specific effect on the levels of alpha- and beta-tubulin mRNAs, causing a five to nine-fold reduction in the steady-state levels of both mRNA populations, whereas the levels of other mRNAs such as gapdh remained unchanged. The reduction in RNA levels caused by vinblastine treatment is mediated by changes in tubulin mRNA half-lives. In an attempt to identify regulatory elements within tubulin mRNAs, plasmids containing luciferase reporter gene associated with 5'-untranslated (UTR), 3'-UTR and part of coding sequence of the tubulin genes were constructed and used for transient DNA transfections of epimastigotes. Determination of luciferase activity in transfected parasites cultured in the presence and absence of vinblastine indicated that sequences located within the alpha-tubulin 3'-UTR and coding region may be involved in modulating the stability of these transcripts in response to changes in the dynamics of T. cruzi microtubules.
da Silva
Rosiane A
RA
Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270-010, Brazil.
Bartholomeu
Daniella C
DC
Teixeira
Santuza M R
SM
D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D006207:Half-Life; D008018:Life Cycle Stages; D049453:Luciferases, Bacterial / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects; D017239:Paclitaxel / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D016053:RNA, Protozoan / Q000032:analysis; D014162:Transfection / Q000379:methods; D014349:Trypanosoma cruzi / Q000166:cytology / Q000187:drug effects / Q000235:genetics*; D014404:Tubulin / Q000235:genetics*; D014747:Vinblastine / Q000494:pharmacology
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000972:Antineoplastic Agents, Phytogenic; D003598:Cytoskeletal Proteins; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D014404:Tubulin; D014747:Vinblastine; D049453:Luciferases, Bacterial; D017239:Paclitaxel