Dataset Information

Off-target responses in the HeLa proteome subsequent to transient plasmid-mediated transfection.

ABSTRACT: Data from ProteomeXchange, PXD ID: PXD001278. File: 140508_SILAC_transfect_HeavyReagent_LightNormal_2.mzml. Published as part of Biochim Biophys Acta. 2014 Nov 5;1854(1):84-9 . From the Abstract: {{i}} Here we have used a SILAC-based proteomic approach to study differentially expressed proteins after transfection of HeLa cells with ECFP vector using a commonly employed non-liposome based transfection reagent, Fugene HD. Whereas the transfection reagent itself mediated minimal effects upon protein expression, 11 proteins were found to be significantly upregulated after transfection, all of which were associated with an interferon type I/II response. The upregulated proteins might potentially inflict major cellular processes such as RNA splicing, chromatin remodeling, post-translational protein modification and cell cycle control. {{/i}}

INSTRUMENT(S): Instrument

ORGANISM(S): Homo_sapiens_viruses, Human_female

DISEASE(S): Not Available

SUBMITTER: Hagen L, et al.

PROVIDER: GPM32320005268 | GPMDB |

REPOSITORIES: GPMDB

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Off-target responses in the HeLa proteome subsequent to transient plasmid-mediated transfection.

Hagen Lars L Sharma Animesh A Aas Per Arne PA Slupphaug Geir G

Biochimica et biophysica acta 20141105 1

Transient transfection of mammalian cells with plasmid expression vectors and chemical transfection reagents is widely used to study protein transport and dynamics as well as phenotypic alterations mediated by the overexpressed protein. Despite the undisputed impact of this technique, surprisingly little is known about the cellular effects mediated by the transfection process per se. Conceivably, off-target effects could have implications upon proteins or processes being studied and understandin ...[more]

PMID: 25448019

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Project description:Malaria parasites must be able to respond quickly to their environment, including during their transmission between mammalian hosts and mosquito vectors. Therefore, before transmission, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. This essential regulatory control requires the orthologues of DDX6 (DOZI) and LSM14 (CITH), which along with ALBA proteins, form a translationally repressive complex in female gametocytes that associates with many of the affected mRNAs. However, while the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, the changes in spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first comparative transcriptomics and proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we find that over 200 transcripts are released for translation soon after fertilization, including those with essential functions for the zygote. However, we also observed that some transcripts remain repressed beyond this point. In addition, we have used TurboID-based proximity proteomics to interrogate the spatial and compositional changes in the DOZI/CITH/ALBA complex across this transmission event. Consistent with recent models of translational control, proteins that associate with either the 5’ or 3’ end of mRNAs are in close proximity to one another during translational repression in female gametocytes and then dissociate upon release of repression in zygotes. These protein interactions in female gametocytes were further validated through structured illumination microscopy and ultrastructure expansion microscopy. Together, these data provide a model for the essential translational control mechanisms used by malaria parasites to promote their efficient transmission.

Project description:Malaria is caused by Plasmodium parasites, which are transmitted via the bites of infected Anopheline mosquitoes. Midgut invasion is a major bottleneck for Plasmodium development inside the mosquito vectors as a rapidly responding immune system recognizes ookinetes and recruits killing factors from the midgut and surrounding tissues, dramatically reducing the population of invading ookinetes before they can successfully traverse the midgut epithelium. Understanding molecular details of the parasite-vector interactions requires precise measurement of nascent protein synthesis in the mosquito during Plasmodium infection. Current expression profiling primarily monitors alterations in steady-state levels of mRNA, but does not address the equally critical issue of whether the proteins encoded by the mRNAs are actually synthesized. In this study, we used sucrose density gradient centrifugation to isolate actively translating Anopheles gambiae mRNAs based upon their association with polyribosomes (polysomes). The proportion of individual gene transcripts associated with polysomes, which is determined by RNA deep sequencing, reflects mRNA translational status. This approach led to identification of 1017 mosquito transcripts that were primarily regulated at the translational level after ingestion of Plasmodium falciparum-infected blood. Caspar, a negative regulator of the NF-kappaB transcription factor Rel2, appears to be substantially activated at the translational levels during Plasmodium infection. In addition, transcripts of Dcr1, Dcr2 and Drosha, which are involved in small RNA biosynthesis, exhibited enhanced associations with polysomes after P. falciparum challenge. This observation suggests that mosquito microRNAs may play an important role in reactions against Plasmodium invasion. We analyzed both total cellular mRNAs and mRNAs that are associated with polysomes to simultaneously monitor transcriptomes and nascent protein synthesis in the mosquito. This approach provides more accurate information regarding the rate of protein synthesis, and identifies some mosquito factors that might have gone unrecognized because expression of these proteins is regulated mainly at the translational level rather than at the transcriptional level after mosquitoes ingest a Plasmodium-infected blood meal.

Dataset Information

Off-target responses in the HeLa proteome subsequent to transient plasmid-mediated transfection.

Publications

Off-target responses in the HeLa proteome subsequent to transient plasmid-mediated transfection.

Similar Datasets

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