Vrielink, A. | bioRxiv Science

bioRxiv2023

Structural analysis of the Sterile alpha motif (SAM) domain of the Arabidopsis mitochondrial tRNA import receptor

Mitochondria are membrane bound organelles of endosymbiotic origin with limited protein coding capacity. As a consequence, the continual import of nuclear-encoded protein and nucleic acids such as DNA and small non-coding RNA is required and essential for maintaining organelle mass, number and activity. As plant mitochondria do not encode all the necessary tRNA types required, the import of cytosolic tRNA is vital for organelle maintenance. Recently, two mitochondrial outer membrane proteins, named Tric1 and Tric2, for tRNA import component, were shown to be involved in the import of cytosolic tRNA. Tric1/2 binds tRNAala via conserved residues in the C-terminal Sterile Alpha Motif (SAM) domain. Here we report the X-ray crystal structure of the Tric1 SAM domain. We identified the ability of the SAM domain to form a helical superstructure with 6 SAM domains per helical turn and key amino acid residues responsible for its formation. We determined that the oligomerization of Tric1 SAM domain was essential for protein function whereby mutation of Gly241 resulted in the disruption of the oligomer and the loss of RNA binding capability in Tric1. Furthermore, complementation of Arabidopsis thaliana Tric1/2 knockout lines with a mutated Tric1 failed to restore the defective plant phenotype suggesting the oligomerization is essential for function in planta. AlphaFold2 structure prediction of the SAM domain and Tric1 support a cyclic hexamer generating a pore of sufficient dimensions to transfer tRNA across the mitochondrial membrane. Our results highlight the importance of oligomerization of Tric1 for protein function.

biochemistry↗

bioRxiv2017

A novel method for single whitefly (Bemisia tabaci) transcriptomes reveals an eleven amino acid deletion in the NusG protein in the bacterial endosymbiont Portiera aleyrodidarum

BackgroundBemisia tabaci species (whiteflies) are the worlds most devastating insect pests within crops in the tropics. They cause billions of dollars (US) of damage each year and are leaving farmers in the developing world food insecure. Understanding the genetic and transcriptomic composition of these insect pests, the viruses they transmit and the microbiota is crucial to sustainable insect and virus management solutions for farmers. Currently, publically available transcriptome data for B. tabaci has been generated from pooled samples (mainly inbred lab colonies) consisting of several individuals because whiteflies are small (approximately 0.2 mm wide and 0.1 mm in height). Pooling individuals can lead to high heterozygosity and skewed representation of the genetic diversity. The ability to extract enough RNA from a single whitefly has remained elusive due to their small size and technology limitations. Therefore, the understanding of whitefly-microbiotad-viral species composition of an individual field-collected whitefly has also remained unknown. In this study, we developed a single whitefly RNA extraction procedure and subsequently successfully sequenced the transcriptome of four individual adult SubdSaharan Africa (SSA1) B. tabaci.\n\nResultsTranscriptome sequencing on individual whiteflies resulted in between 39-42 million raw reads. De novo assembly of trimmed reads yielded between 65,000-162,000 transcripts across all four B. tabaci transcriptomes. In addition, Bayesian phylogenetic analysis of mitochondrion cytochrome I oxidase (mtCOI) grouped the four whiteflies within the SSA1 clade. BLAST searches on assembled transcripts within the four individual transcriptomes identified five endosymbionts; the primary endosymbiont Portiera,aleyrodidarum and four secondary endosymbionts: Arsenophonus, Wolbachia, Rickettsia, and Cardinium spp. These five endosymbionts were predominant across all four SSA1 B. tabaci study samples with prevalence levels of between 54.1d75%. Nucleotide and amino acid sequence alignments of the NusG gene of P. aleyrodidarum for the SSA1 B. tabaci transcriptomes of samples WF2 and WF2b revealed an eleven amino acid residue deletion that was absent in samples WF1 and WF2a. Comparison of the protein structure of the NusG protein from P. aleyrodidarum in SSA1 with known NusG structures showed the deletion resulted in a shorter D loop. Although NusG is key in regulating of transcription elongation, it is believed that the shortening of the loop region in the N-terminal domain is unlikely to affect transcription termination. Therefore, the effect of variability in this region across species is unknown.\n\nConclusionIn this study, we optimised a single whitefly high quality RNA extraction procedure and successfully carried out individual whitefly transcriptome sequencing on adult B. tabaci whiteflies. This enabled the detection of unique genetic differences in the NusG genes of the primary endosymbiont P. aleyrodidarum in four field-collected SSA1 whiteflies that may not have been detected using lab-pooled B. tabaci isolines. The use of field-collected specimens means that both time and money will be saved in future studies using single whitefly transcriptomes in monitoring vector and viral interactions. In addition, the methods we have developed here are applicable to any small organism where RNA quantity has limited transcriptome studies.

genetics↗