RESUMO
ISWI chromatin remodelers are highly conserved in eukaryotes and are important for the assembly and spacing of nucleosomes, thereby controlling transcription initiation and elongation. ISWI is typically associated with different subunits, forming specialized complexes with discrete functions. In the unicellular parasite Trypanosoma brucei, which causes African sleeping sickness, TbISWI down-regulates RNA polymerase I (Pol I)-transcribed variant surface glycoprotein (VSG) gene expression sites (ESs), which are monoallelically expressed. Here, we use tandem affinity purification to determine the interacting partners of TbISWI. We identify three proteins that do not show significant homology with known ISWI-associated partners. Surprisingly, one of these is nucleoplasmin-like protein (NLP), which we had previously shown to play a role in ES control. In addition, we identify two novel ISWI partners, regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP), both containing protein motifs typically found on chromatin proteins. Knockdown of RCCP or FYRP in bloodstream form T. brucei results in derepression of silent variant surface glycoprotein ESs, as had previously been shown for TbISWI and NLP. All four proteins are expressed and interact with each other in both major life cycle stages and show similar distributions at Pol I-transcribed loci. They are also found at Pol II strand switch regions as determined with ChIP. ISWI, NLP, RCCP, and FYRP therefore appear to form a single major ISWI complex in T. brucei (TbIC). This reduced complexity of ISWI regulation and the presence of novel ISWI partners highlights the early divergence of trypanosomes in evolution.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Trypanosoma brucei brucei/genética , Animais , Técnicas de Silenciamento de Genes , Genes de Protozoários , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Transcrição Gênica , Glicoproteínas Variantes de Superfície de Trypanosoma/química , Glicoproteínas Variantes de Superfície de Trypanosoma/genéticaRESUMO
CCCH zinc finger proteins (ZC3Hs) are a novel class of RNA-binding protein involved in post-transcriptional mechanisms controlling gene expression. We show TbZC3H20 from Trypanosoma brucei, the causative agent of sleeping sickness and other diseases, stabilizes two developmentally regulated transcripts encoding a mitochondrial carrier protein (MCP12) and trans-sialidase (TS-like E). TbZC3H20 is shown to be an RNA-binding protein that is enriched in insect procyclic form T. brucei and is the first ZC3H discovered controlling gene expression through modulating mRNA abundance in trypanosomes. Previous studies have demonstrated that RNA recognition motif-containing and PUF family RNA-binding proteins can control gene expression by stabilizing specific target mRNA levels. This work is the first to describe a ZC3H stabilizing rather than destabilizing target mRNAs as a regulatory mechanism and the first report of a ZC3H regulating a gene encoding a mitochondrial protein. This suggests a broader role for ZC3Hs in post-transcriptional regulation of gene expression than previously thought.
Assuntos
Proteínas de Protozoários/metabolismo , Proteínas de Ligação a RNA/metabolismo , Trypanosoma brucei brucei/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Proteínas de Protozoários/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA de Protozoário/biossíntese , RNA de Protozoário/genética , Proteínas de Ligação a RNA/genética , Trypanosoma brucei brucei/genética , Dedos de Zinco/fisiologiaRESUMO
Individuals with lymphogranuloma venereum (LGV), caused by Chlamydia trachomatis serovar L2, are commonly co-infected with human immunodeficiency virus type 1 (HIV-1), for reasons that remain unknown. One hypothesis is that a biological synergy exists between the two pathogens. We tested this by characterising for the first time in vitro C. trachomatis L2 replication in the presence of HIV-1. The human epithelial cell-line, MAGI P4R5 was infected with C. trachomatis L2 and HIV-1 (MN strain). Co-infected cultures contained fewer and larger chlamydial inclusions, but the inclusions did not contain morphologically aberrant organisms. C. trachomatis remained infectious in the presence of HIV-1 and showed neither an alteration in genome accumulation, nor in the acumulation of ompA, euo or unprocessed 16S rRNA transcripts. However, omcB was slightly elevated. Taken together, these data indicate that HIV-1 co-infection did not significantly alter C. trachomatis replication and the association between HIV-1 and LGV is likely due to other factors that require further investigation. The fewer, larger inclusions observed in co-infected cultures probably result from the fusion of multiple inclusions in HIV-1 induced syncytia and indicate that C. trachomatis-host-cell interactions continue to function, despite considerable host-cell re-modelling.