| Abstract |
“RNA Silencing” constitutes a molecular mechanism that is mediated by a variety of protein complexes, which suppress specific nucleic acids, guided by antisense small RNAs ~20- 33 nt long. “RNA Silencing” is not an identical mechanism throughout organisms. In each and one of them, shows diverse and unique evolutionary specificities.
Except from all the proteins and RNA molecules, and in general, all the factors that act positively mediating the molecular mechanism, there is a variety of factors that act negatively/ regulatory to “RNA Silencing”. These factors are divided into exogenous and endogenous. The exogenous factors include, mainly, viral proteins, whereas the endogenous include proteins of the organism itself. Among these endogenous factors a plant protein is also included, ERL1 (ERI1-LIKE 1), the plant orthologue of ERI1 (ENHANCED RNAi 1), a 3’→ 5’ exonuclease, which has been identified from genetic screens of C. elegans mutants showing hypersensitivity to dsRNA. Onwards, different orthologues of ERI1 have been identified in various eukaryotes, such as ERI1 from M. musculus (MmERI1), D. melanogaster (Snipper), N. crassa (QIP), H. sapiens (Thex1/3’hExo), as well as from plants.
ERI1 proteins possess an ERI 1_3’hExo domain with RNase H activity, and an aminoterminally localized SAP (SAF A/B/ Acinus/ PIAS) domain, which confers RNA/ DNA binding capacity. The presence or absence of the SAP domain, classifies ERI1 proteins into Group Ι or Group ΙΙ ERI1s, respectively. The ERL1 (ERI1- LIKE 1) of A. thaliana (AtERL1) possesses, as well, an aminoterminal signal peptide that guides the protein into the chloroplast, a subcellular department known as an “RNA Silencing” free organelle.
From previous laboratory results, it seems quite promising that ERL1 may facilitate different molecular pathways than the regulation of “RNA Silencing”, concerning the fact that ERL1 is unable to affect the expression of the GFP transgene, due to its subcellular localization, and the accumulation of 5S rRNA molecules 2 nt longer at their 3’- ends from plants expressing ERL1 ectopically.
In the context of this MSc project, motivated by the available laboratory results and recent publications concerning the involvement of few of the animal ERI1 orthologues to the maturation of the 3’- end of 5.8S rRNA, a variety of experiments were undertaken, in order to validate the function of ERL1 (ERI1- LIKE 1) protein of A. thaliana to different RNA substrates.
AtERL1 was purified by affinity chromatography under native conditions, concurrently with the native purification of it’s orthologous from M. musculus (MmERI1). MmERI1 in vitro reactions were going to be utilized as positive control reactions, for the investigation of putative involvements of AtERL1 to the 3’- end maturation of the chloroplastic 5S rRNA, similar to MmERI1 function upon 5.8S rRNA. Unfortunately, due to degradation problems during the repetition of the experiments of Ansel et al. 2008, it was not feasible to complete the above experiment.
Additionally, we have attempted to detect ERL1, using Western blotting, in various N. benthamiana wild type (WT) samples. However, after a variety of different blottings, even in cases that extracts of purified chloroplasts were utilized, the detection of WT ERL1 was unsuccessful.
Finally, ERL1 protein levels have been detected in different N. benthamiana transgenic lines that constitutively overexpress the AtERL1. Specifically, the transgenic lines “Mosaic”, “Bleach” and «No- Phenotype», due to their phenotypic characteristics of green and white mosaic spots on their leaves, completely bleached leaves, and without any chlorotic phenotype, respectively. Although “Bleach” and “Mosaic” lines show approximately the same ERL1 protein profile, in the case of the “No- Phenotype” line, the protein levels of ERL1 are severely lowered, approximately half the amount. Concerning this, it is highly prominent to speculate that lower ERL1 expression may not confer any characteristically chlorotic phenotype in «No- Phenotype» N. benthamiana transgenic lines.
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