Final Report Summary - DROSEJCINRNAREG (Towards a complete understanding of the roles of the Exon Junction Complex in Drosophila: Identification and functional characterization of the RNA targets and protein partners of the EJC)
An important aspect of mRNA regulation in living cells is nuclear mRNA packaging. The assembly of RNA and RNA binding proteins (RBPs) into messenger ribonucleoprotein complexes (mRNPs) allows information on nuclear events, such as splicing or polyadenylation, to be transmitted to the cytoplasm. Some of the RBPs can be therefore considered as landmarks of nuclear processing events on cytoplasmic mRNAs. One of these protein landmarks is the multi-protein Exon Junction Complex (EJC). The EJC core consists of four conserved subunits, the DEAD box helicase eIF4AIII, Mago nashi, Y14 and Barentsz (Btz) which are recruited to nuclear mRNAs 20-24 nt upstream of exon-exon junctions upon splicing and thereby form the holo-EJC complex. Whereas in mammals, EJC is a central component of RNA surveillance, marking incorrectly spliced mRNAs in the cytoplasm for non-sense-mediated decay (NMD), in Drosophila, the EJC has been shown to be essential for transport and localization of the maternal oskar mRNA to the posterior of the developing oocyte, a prerequisite for the formation of germ cells in the developing embryo.
The main objective of my project was to investigate the molecular engagement of EJC in the regulation of mRNA biogenesis using the fruitfly Drosophila melanogaster as a model. This goal was to be achieved by two means: first to define the EJC associated transcriptome in the living organism, second to investigate its associated proteome at detected mRNAs which should provide insights into EJC function and regulation.
During the project period:
i) I established transgenic flies co-expressing EJC subunits, differentially tagged with specific epitopes, for biochemical purification of individual EJC subunits.
ii) I designed and set up an assay in Drosophila S2 tissue cell culture to investigate how the EJC-disassembly factor PYM exerts its activity in the cell cytoplasm.
iii) I established a working protocol allowing the isolation of holo-EJC complexes bound to their mRNA targets, from naïve, translationally competent cell cytoplasm of adult flies.
iv) I set up RNAseI protection on purified holo-EJCs which in combination with an accelerated cDNA library preparation protocol and comabination genome wide Next Generation Sequencing I utilize to define the EJC associated transcriptome in Drosophila.
I revealed in collaboration with colleagues in the host group that the regulation of EJC stability in Drosophila cytoplasm differs from mammals. Investigating the function of the EJC disassembly factor PYM in this process, we found that its activity in Drosophila is dispensable for the viability and cellular homeostasis of the fly. In contrast to mammals, Drosophila PYM does not bind ribosomes. I found instead, that the activity of Drosophila PYM is regulated, suggesting that PYM in the fly is maintained in the cell cytoplasm in a defined relation of its dormant and active state. Overexpression in fly oocytes of an engineered PYM isoform that is constitutively active and depletes EJCs efficiently from mRNAs, suggested that except for oskar, for most other mRNAs in the fly ooplasm, EJC-RNA association does not exert any function. My data contributed to a recent publication in the journal PLoS Genetics, to which I contributed as second author.
Confirming the previous finding, I observed in genome wide sequencing results of the EJC associated transcriptome, that in adult flies only a subset of cytoplasmic mRNAs are stably associated with the EJC. Interestingly, a great portion of the detected transcripts, do not show a homogenous, but rather a differential distribution of EJC deposition along exon-exon junctions. A prominent representative in this class of transcripts is the oskar mRNA. I found that in vivo, EJC primarily bound to first exon-exon junction in the oskar mRNA, but is hardly present if not completely absent at its second and third junction. This is of importance since the host group showed in the past that nuclear splicing of at the first junction is a pre-requisite for posterior localization of oskar mRNA in the oocyte. In contrast, the host group showed that in vitro all oskar exon-exon junctions are bound with EJCs upon splicing in vitro. This suggests that the distribution of isolated EJCs along bound transcripts in vivo may differ significantly from what was observed in the past based on EJC binding in vitro. Currently investigations into this are still ongoing and will be finalized in the next 6 months. In collaboration with the laboratory of Jernej Ule, I am currently developing; a pipeline for data mining my sequencing results, allowing us an automated evaluation of further EJC sequencing approaches. Ultimately, this analysis will generate a transcriptome wide, comprehensive catalogue of EJC binding sites, their distribution and with this, the mode of posttranscriptional regulation of the associated transcripts will be established.
Once published, my results will be of special importance for the field, since they will provide novel and profound insights into the complex interplay of co-transcriptional and posttranscriptional regulation of gene expression in living organisms. This will widen our knowledge of how posttranscriptional control of gene-expression may impact cell polarity, and consequently how it impacts cellular differentiation and proliferation, during animal development, organogenesis and tissue homeostasis, and finally cancer development.
The main objective of my project was to investigate the molecular engagement of EJC in the regulation of mRNA biogenesis using the fruitfly Drosophila melanogaster as a model. This goal was to be achieved by two means: first to define the EJC associated transcriptome in the living organism, second to investigate its associated proteome at detected mRNAs which should provide insights into EJC function and regulation.
During the project period:
i) I established transgenic flies co-expressing EJC subunits, differentially tagged with specific epitopes, for biochemical purification of individual EJC subunits.
ii) I designed and set up an assay in Drosophila S2 tissue cell culture to investigate how the EJC-disassembly factor PYM exerts its activity in the cell cytoplasm.
iii) I established a working protocol allowing the isolation of holo-EJC complexes bound to their mRNA targets, from naïve, translationally competent cell cytoplasm of adult flies.
iv) I set up RNAseI protection on purified holo-EJCs which in combination with an accelerated cDNA library preparation protocol and comabination genome wide Next Generation Sequencing I utilize to define the EJC associated transcriptome in Drosophila.
I revealed in collaboration with colleagues in the host group that the regulation of EJC stability in Drosophila cytoplasm differs from mammals. Investigating the function of the EJC disassembly factor PYM in this process, we found that its activity in Drosophila is dispensable for the viability and cellular homeostasis of the fly. In contrast to mammals, Drosophila PYM does not bind ribosomes. I found instead, that the activity of Drosophila PYM is regulated, suggesting that PYM in the fly is maintained in the cell cytoplasm in a defined relation of its dormant and active state. Overexpression in fly oocytes of an engineered PYM isoform that is constitutively active and depletes EJCs efficiently from mRNAs, suggested that except for oskar, for most other mRNAs in the fly ooplasm, EJC-RNA association does not exert any function. My data contributed to a recent publication in the journal PLoS Genetics, to which I contributed as second author.
Confirming the previous finding, I observed in genome wide sequencing results of the EJC associated transcriptome, that in adult flies only a subset of cytoplasmic mRNAs are stably associated with the EJC. Interestingly, a great portion of the detected transcripts, do not show a homogenous, but rather a differential distribution of EJC deposition along exon-exon junctions. A prominent representative in this class of transcripts is the oskar mRNA. I found that in vivo, EJC primarily bound to first exon-exon junction in the oskar mRNA, but is hardly present if not completely absent at its second and third junction. This is of importance since the host group showed in the past that nuclear splicing of at the first junction is a pre-requisite for posterior localization of oskar mRNA in the oocyte. In contrast, the host group showed that in vitro all oskar exon-exon junctions are bound with EJCs upon splicing in vitro. This suggests that the distribution of isolated EJCs along bound transcripts in vivo may differ significantly from what was observed in the past based on EJC binding in vitro. Currently investigations into this are still ongoing and will be finalized in the next 6 months. In collaboration with the laboratory of Jernej Ule, I am currently developing; a pipeline for data mining my sequencing results, allowing us an automated evaluation of further EJC sequencing approaches. Ultimately, this analysis will generate a transcriptome wide, comprehensive catalogue of EJC binding sites, their distribution and with this, the mode of posttranscriptional regulation of the associated transcripts will be established.
Once published, my results will be of special importance for the field, since they will provide novel and profound insights into the complex interplay of co-transcriptional and posttranscriptional regulation of gene expression in living organisms. This will widen our knowledge of how posttranscriptional control of gene-expression may impact cell polarity, and consequently how it impacts cellular differentiation and proliferation, during animal development, organogenesis and tissue homeostasis, and finally cancer development.