Analysis of a putative translation initiation factor of mitoribosomes in Arabidopsis thaliana

Abstract

Mitochondria play a pivotal role in eukaryotic energy metabolism. Like mitochondria across most eukaryotic species, plant mitochondria contain their own genomes and protein synthesis machinery. However, many plant mitochondrial key proteins and processes remain poorly understood. An example of this is the plant mitochondrial translation initiation. Although the cryoelectron microscopy (EM) structure of the plant mitoribosome has recently been revealed, translation initiation in plant mitochondria is completely unknown. A known type of translation initiation involves the Shine-Dalgarno (SD) sequences in the 5’ untranslated regions (5’UTRs) of the mRNAs in prokaryotes and archaea. Here, specific motifs occur in the 5’UTRs, which aid in recruiting the ribosome. In plant mitochondrial mRNA, such motifs have never been reported thus far. Previous studies showed that mTRAN1 is a part of the mitoribosomal small subunit (mtSSU) and is suggested to be involved in translation initiation. Since mTRAN1 is likely to belong to a protein family, which is known to interact with RNA, mTRAN1 was suspected to interact with mRNA. In this work, a potential mechanism of mitochondrial translation initiation using the novel plant-specific translation initiation factor mTRAN1 and RNA motifs was explored. To predict the capacity of potential RNA-binding, mTRAN1 protein structure was redicted using multiple structure prediction algorithms. Then, RNA electromobility shift assays (REMSAs) were performed to analyze the interaction between mTRAN1 and RNA oligonucleotides. Hereby, REMSAs showed that mTRAN1 binds to the 5’UTRs of mitochondrial mRNAs, and the presence of A/U-rich regions enhances the binding significantly. To further study the importance of mTRAN1 for mRNA-mitoribosome interaction, crude mitoribosomal fractions were isolated from the wildtype (wt) plants and mtran double knockout mutants. Said mitoribosomal fractions of different genotypes were then analyzed via REMSA as well, to investigate whether the absence of mTRAN proteins influences the mRNA:mitoribosome interaction. However, the large size of the mitoribosomes (~10 MDa) caused complications for REMSAs that could not be solved in the span of this work. In conclusion, the results of this project suggest that the mechanisms of mitoribosomal translation initiation takes place in a very different way in comparison with those of other eukaryotes.

Popular Abstract

Plants are essential for most life on earth, providing more than 80% of the food consumed by humans and being the primary source of nutrition for animals, including livestock (Rizzo et al., 2021). Therefore, it is essential that we understand every aspect of plant physiology, to be able to counteract threats to plant health such as pests or changing environmental conditions.
One core process in all living organisms, including plants, is protein biosynthesis. Proteins are the large biomolecules facilitating most functions in and around cells. Proteins are made up of long amino acid chains, which are encoded by DNA. To get a protein from a DNA sequence, the DNA is first transcribed into messenger RNA (mRNA), which is then translated into amino acids by the ribosome. This happens inside cells; a separate protein synthesis machinery and DNA are also present in mitochondria. The mitochondrion is a cellular organelle that is needed to convert different forms of chemical energy into the universal energy currency of the cell called adenosine-triphosphate (ATP). Mitochondria generate ATP through respiration and can then provide energy to many other processes in the plant. Although mitochondria have a pivotal role in plant cells, to date, we do not know how plant mitochondrial protein translation works.
In this work, a group of proteins called mitochondrial TRANslation factors (mTRAN) is studied, which can only be found in land plants. As a model organism, mouse-ear cress (Arabidopsis thaliana) was chosen. If these proteins are absent in the plant (e.g., due to mutations), the plant is significantly smaller and shows many signs of metabolic dysfunction. In some cases, the absence of these proteins is lethal for the plant. This leads to the conclusion that the mTRAN proteins fulfil an important function inside the plant. However, so far mTRAN proteins have only been poorly characterized. In recent studies, mTRAN proteins have been found to be part of the mitochondrial ribosome. It belongs to a protein family which is known to interact with RNA. They are involved in mitochondrial translation initiation (Tran et al., in revision). The translation initiating step is important for protein synthesis and has severe consequences if impaired. Translation initiation can happen in many ways, depending on the organism. In bacteria, for example, certain RNA sequences attract the ribosome which initiates the translation.

Here, we studied whether an mTRAN protein can bind to RNA and is responsible for recruiting mRNA to the ribosome. By predicting the 3D structure of an mTRAN protein, it could be confirmed that it very likely belongs to a protein family that is known to interact with RNA. It could be shown that the mTRAN protein does not only bind RNA but also has a preference for certain RNA sequences that are rich in adenosine (A) and uracil (U) (two of the four RNA building blocks). Future studies must show whether the affinity of mTRAN1 to these sequences directly causes translation initiation or if it has a more passive effect on it. Nonetheless, this study was able to uncover many interesting characteristics of the essential plant protein mTRAN and give a new lead to further exploring plant mitochondrial translation.

• author: Schmitt, Vivian
• supervisor: Urban Johanson; Olivier Van Aken
• organization: Department of Chemistry
• course: KEMR30 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Arabidopsis thaliana, Mitochondrial Ribosomes, Translation initiation, Biochemistry
• language: English
• id: 9095116
• date added to LUP: 2022-08-01 13:19:31
• date last changed: 2022-08-01 13:19:31