Actively transcribing RNA polymerase II concentrates on spliced leader genes in the nucleus of Trypanosoma cruzi.
RNA polymerase II of trypanosomes, early diverging eukaryotes, transcribes long polycistronic messages, which are not capped but are processed by trans-splicing and polyadenylation to form mature mRNAs. The same RNA polymerase II also transcribes the genes coding for the spliced leader RNA, which are capped, exported to the cytoplasm, processed, and reimported into the nucleus before they are used as splicing donors to form mRNAs from pre-mRNA polycistronic transcripts. As pre-mRNA and spliced leader transcription events appear to be uncoupled, we studied how the RNA polymerase II is distributed in the nucleus of Trypanosoma cruzi. Using specific antibodies to the T. cruzi RNA polymerase II unique carboxy-terminal domain, we demonstrated that large amounts of the enzyme are found concentrated in a domain close to the parasite nucleolus and containing the spliced leader genes. The remaining RNA polymerase II is diffusely distributed in the nucleoplasm. The spliced leader-associated RNA polymerase II localization is dependent on the cell transcriptional state. It disperses when transcription is blocked by alpha-amanitin and actinomycin D. Tubulin genes are excluded from this domain, suggesting that it may exclusively be the transcriptional site of spliced leader genes. Trypomastigote forms of the parasite, which have reduced spliced leader transcription, show less RNA polymerase II labeling, and the spliced leader genes are more dispersed in the nucleoplasm. These results provide strong evidences that transcription of spliced leader RNAs occurs in a particular domain in the T. cruzi nucleus.
Dossin
Fernando de Macedo
Fde M
Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, R. Botucatu 862 8 andar, 04023-062 São Paulo, Brazil. sergio@ecb.epm.br.
Schenkman
Sergio
S
D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry / Q000201:enzymology*; D015800:Protozoan Proteins / Q000032:analysis / Q000378:metabolism*; D012319:RNA Polymerase II / Q000032:analysis / Q000378:metabolism*; D020038:RNA, Spliced Leader / Q000235:genetics*; D014158:Transcription, Genetic; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics*; D014404:Tubulin / Q000235:genetics
D015800:Protozoan Proteins; D020038:RNA, Spliced Leader; D014404:Tubulin; D012319:RNA Polymerase II