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Identifier

000402932

Title

Enhanced RNA interference-1-like-1 : το φυτικό ομόλογο των ERI-1 πρωτεϊνών συμμετέχει στην κατεργασία των ριβοσωμικών μεταγραφών του χλωροπλάστη

Alternative Title

Enhanced RNA interference-1-like-1: the ERI-1 plant homologue participates in the processing of chloroplastic ribosomal transcripts

Author

Μέρμηγκα, Γλυκερία Γ.

Thesis advisor

Καλαντίδης, Κρίτων

Reviewer

Βόντας, Ιωάννης
Δελιδάκης, Χρήστος
Καμπράνης, Σωτήριος
Κοτζαμπάσης, Κυριάκος
Τζαμαρίας, Δημήτριος
Τσαγρή, Ευθυμία

Abstract

Ribonucleases are involved in a variety of processes in all organisms, from viruses to higher eukaryotes performing essential roles in RNA transcription termination, maturation, editing, splicing and RNA silencing. Depending on their mode of action and some intrinsic characteristics they are grouped in several families one of which is the subfamily Enhancer of RNA interference-1 (ERI-1). Proteins of this group are 3’-5’ exoribonucleases belonging to the larger family of DEDDh nucleases in which the bacterial oligoribonuclease and RNase T are also members. ERI-1, a protein conserved in eukaryotes from the nematode to mouse, humans, drosophila, fission yeast and plants participates in various processes. These include degradation of siRNAs thus acting as suppressors of RNAi 3’ end trimming of the short 5.8S rRNA and degradation of histone mRNA. Arabidopsis thaliana Eri-1 homologue (ERI-1-Like-1, ERIL1) is targeted to the chloroplast. Nicotiana benthamiana transgenic lines overexpressing AtERIL1 present chlorotic phenotypes with abnormal plastid anatomy which resemble proplastids. Molecular analysis of these transgenic lines revealed low levels of chloroplasrtic transcripts compared to wild type plants. Aim of this work was to further study the role of Eri-1 plant homologue in the model plants A. thaliana and N. benthamiana. Initially, and due to the presence in N. benthamiana of two genetic loci coding for ERIL1, sequencing of the different transcripts was conducted which revealed that two differentially spliced forms arise from one of the two paralogue loci. In order to investigate the role of alternative splicing in subcellular localization of the protein, the genetic locus was fused to the reporter gene Green Fluorescent Protein and the construct was overexpressed in N. benthamiana leaves. Confocal analysis on protoplasts prepared from these tissues revealed its chloroplastic localization. The above result was also verified by western blot of chloroplastic extracts using an ERIL1 specific antibody. Next, the transgenic lines of both model plants with reduced expression levels where characterized by northern and western blot. The suppression lines had reduced chlorophyll content while transmission electron microscopy on leaf section from these N.benthamiana lines revealed abnormal chloroplast anatomy compared to wild type plants. In order to investigate a probable impact of ERIL1 on the chloroplast transcriptome, northern analysis was conducted from total RNA extracted from lines that overexpress and suppress ERIL1. The analysis showed a pronounced effect on the ribosomal RNAs suggesting an impact on the maturation process of these transcripts. Additionally to the above, immunoprecipitaion assays implied a role of ERIL1 also on some other chloroplastic transcripts. Transient overexpression of ERIL1 on fully expanded leaves followed by northern blot analysis revealed a role in the degradation of chloroplastic 4.5S rRNA. Lastly, the role of ERIL1 on RNA silencing was studied. From these experiments no apparent impact of ERIL1 in the accumulation of exogenous and viroid derived siRNAs could be concluded with the exception of specific miRNAs which overaccumulated in the transgenic plants that overexpressed the transgene. Altogether it can be concluded that ERIL1 participates in the maturation of the chloroplastic ribosomal RNAs and in the degradation of 4.5S rRNA while it has no effect on the RNA silencing pathways studied in this work.

Language

Greek

Subject

Chloroplast

Exoribonuclease

RNA silencing

RNA σίγηση

Εξωριβονουκλεάση

Issue date

2016-09-22

Collection

School/Department--School of Sciences and Engineering--Department of Biology--Doctoral theses