Abstract
BackgroundSplicing and alternate splicing are the two key biological processes that result in the generation of diverse transcript and protein isoforms in Plasmodium falciparum as well as in other eukaryotic organisms. Not much is known about the organization of splicing machinery and mechanisms in human malaria parasite. Present study reports the organization and assembly of Plasmodium spliceosome Sm core complex.MethodsPresence of all the seven Plasmodium Sm-like proteins in the intra-erythrocytic stages was assessed based on the protein(s) expression analysis using immuno-localization and western blotting. Localization/co-localization studies were performed by immunofluorescence analysis on thin parasite smear using laser scanning confocal microscope. Interaction studies were carried out using yeast two-hybrid analysis and validated by in vitro pull-down assays. PfPRMT5 (arginine methyl transferase) and PfSmD1 interaction analysis was performed by pull-down assays and the interacting proteins were identified by MALDI-TOF spectrometry.ResultsPfSm proteins are expressed at asexual blood stages of the parasite and show nucleo-cytoplasmic localization. Protein-protein interaction studies showed that PfSm proteins form a heptameric complex, typical of spliceosome core complex as shown in humans. Interaction of PfSMN (survival of motor neuron, tudor domain containing protein) or PfTu-TSN (Tudor domain of Tudor Staphylococcal nuclease) with PfSmD1 proteins was found to be methylation dependent. Co-localization by immunofluorescence and co-immunoprecipitation studies suggested an association between PfPRMT5 and PfSmD1, indicating the role of arginine methylation in assembly of Plasmodium spliceosome complex.ConclusionsPlasmodium Sm-like proteins form a heptameric ring-like structure, although the arrangement of PfSm proteins slightly differs from human splicing machinery. The data shows the interaction of PfSMN with PfSmD1 and this interaction is found to be methylation dependent. PfPRMT5 probably exists as a part of methylosome complex that may function in the cytoplasmic assembly of Sm proteins at asexual blood stages of P. falciparum.
Highlights
Splicing and alternate splicing are the two key biological processes that result in the generation of diverse transcript and protein isoforms in Plasmodium falciparum as well as in other eukaryotic organisms
The phylogenetic analysis revealed that Plasmodium Sm and Lsm proteins form a distinct subgroup with yeast and human Sm proteins, which is divergent from the Trypanosoma group (Figure 1B and see Additional file 2: Figure S1)
To know whether a similar complex is involved in Plasmodium spliceosome asembly, co-localization and coimmunoprecipitation studies using anti-PfSmD1 and anti-PfPRMT5 antibodies were performed and the results clearly demonstrated an association between PfPRMT5 and PfSmD1
Summary
Splicing and alternate splicing are the two key biological processes that result in the generation of diverse transcript and protein isoforms in Plasmodium falciparum as well as in other eukaryotic organisms. Present study reports the organization and assembly of Plasmodium spliceosome Sm core complex. Splicing is a fundamental process present in most eukaryotic cells, including Plasmodium that excises introns from its precursor mRNA (pre-mRNA) to generate mature mRNA [2]. Pre-mRNA splicing is catalysed by the spliceosome, which consists of U1, U2, U4/U6 and U5 snRNAs and numerous splicing-related proteins [3]. SnRNAs associate with Sm proteins in cytoplasm and the assembled snRNPs are subsequently imported to the nucleus where pre-mRNA splicing occurs [18,19,20]. The SMN complex stringently recognizes snRNAs and the RNA-binding Sm proteins and facilitates snRNP assembly [21]
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