For example, prevent the interaction of this protein with components of TREX from S.cerevisiae. Such an example has been recently reported by Va��zquez and colleagues. TcU2AF35, which is involved in the initial steps of trans-splicing in T. cruzi, is able to functionally complement yeast cells when only two aminoacid residues are modified. This demonstrates the importance of conserved residues for functional substitution of ortholog proteins between different organisms. Another possibility might be the development of novel components in the T. cruzi pathway, where specific factors would be necessary for functional activity of TcSub2. Like Sub2 and UAP56, TcSub2 is exclusively nuclear and is dispersed in loci all over the nuclei, and it is also present at the periphery of nucleolus, excluding the fibrillar center of nucleolus. The ultrastructural immunocytochemical assays showed that TcSub2 is concentrated in Catharanthine sulfate non-dense chromatin areas and grouped mainly at the interface between dense and non-dense chromatin. This speckled pattern of distribution is usually associated with mRNA transcription, processing, and nucleocytoplasmatic export. This distribution pattern is LOUREIRIN-B similar to human and arthropod TcSub2 homologues which are localized on the periphery of dense chromatin domains, termed interchromatin granule clusters. These clusters contain mainly proteins related to mRNA processing, especially SC-35, and are closely related with perichromatic fibrils, where mRNA transcription occurs. Many studies have demonstrated that nascent mRNAs are deposited in these interchromatin spaces. A similar distribution pattern has been observed in trypanosomatids, at the interface between dense and non-dense chromatin for bromodomain factors and acetylated histones. Recent investigations show that these histone modifications can serve as indicators of regions for initiation of RNA Pol II transcription. Based on our findings for TcSub2 localization, we decided to investigate the relationship of TcSub2 and active transcription sites using BrUTP incorporation followed by immunocolocalization. This approach has been successfully used to observe the association of RNA Pol II transcription sites with proteins such as Hrp59 and Hrp65, and hnRNPs in C. tentans. It has also been used to define the localization of RNA Pol II transcription sites in T. cruzi. Our results demonstrated that TcSub2 also colocalizes with nascent RNAs �� specifically 
with those transcribed by RNA pol II responsible for mRNA, snRNA and spliced-leader transcription. Blocking of RNA pol II activity with a-amanitin abolished the transcription of nascent RNAs, resulting in RNAs transcribed only by RNA pol I. Under these conditions, we observed the absence of colocalization with TcSub2, indicating that TcSub2 might be associated with RNA pol II, but not with RNA pol I transcription. Besides, the protein does not localize with SL-RNA, reinforcing that the protein is related to mRNA transcription sites. The association of TcSub2 with RNA pol II transcription sites strongly suggests that TcSub2 is functionally similar to homolog proteins in other eukaryotes. However we are still unable to confirm where TcSub2 is involved in the pathway of nuclear mRNA metabolism in this parasite. We also observed uncorrelated peaks of TcSub2 and BrRNA, suggesting that RNA pol II transcription sites are not the only nuclear domain associated with TcSub2. We speculate that the partial association with nascent mRNAs can be explained by the dynamics of interaction between different events in the cell.