Novel strategies to induce male sterility and restore fertility in Brassicaceae crops

Author(s):  
Oren Ostersetzer-Biran ◽  
Jeffrey Mower

Abstract Mitochondria are the site of respiration and numerous other metabolic processes required for plant growth and development. Increased demands for metabolic energy are observed during different stages in the plants life cycle, but are particularly ample during germination and reproductive organ development. These activities are dependent upon the tight regulation of the expression and accumulation of various organellar proteins. Plant mitochondria contain their own genomes (mtDNA), which encode for rRNAs, tRNAs and some mitochondrial proteins. Although all mitochondria have probably evolved from a common alpha-proteobacterial ancestor, notable genomic reorganizations have occurred in the mtDNAs of different eukaryotic lineages. Plant mtDNAs are notably larger and more variable in size (ranging from 70~11,000 kbp in size) than the mrDNAs in higher animals (16~19 kbp). Another unique feature of plant mitochondria includes the presence of both circular and linear DNA fragments, which undergo intra- and intermolecular recombination. DNA-seq data indicate that such recombination events result with diverged mitochondrial genome configurations, even within a single plant species. One common plant phenotype that emerges as a consequence of altered mtDNA configuration is cytoplasmic male sterility CMS (i.e. reduced production of functional pollen). The maternally-inherited male sterility phenotype is highly valuable agriculturally. CMS forces the production of F1 hybrids, particularly in predominantly self-pollinating crops, resulting in enhanced crop growth and productivity through heterosis (i.e. hybrid vigor or outbreeding enhancement). CMS lines have been implemented in some cereal and vegetables, but most crops still lack a CMS system. This work focuses on the analysis of the molecular basis of CMS. We also aim to induce nuclear or organellar induced male-sterility in plants, and to develop a novel approach for fertility restoration. Our work focuses on Brassicaceae, a large family of flowering plants that includes Arabidopsis thaliana, a key model organism in plant sciences, as well as many crops of major economic importance (e.g., broccoli, cauliflower, cabbage, and various seeds for oil production). In spite of the genomic rearrangements in the mtDNAs of plants, the number of genes and the coding sequences are conserved among different mtDNAs in angiosperms (i.e. ~60 genes encoding different tRNAs, rRNAs, ribosomal proteins and subunits of the respiratory system). Yet, in addition to the known genes, plant mtDNAs also harbor numerous ORFs, most of which are not conserved among species and are currently of unknown function. Remarkably, and relevant to our study, CMS in plants is primarily associated with the expression of novel chimericORFs, which likely derive from recombination events within the mtDNAs. Whereas the CMS loci are localized to the mtDNAs, the factors that restore fertility (Rfs) are identified as nuclear-encoded RNA-binding proteins. Interestingly, nearly all of the Rf’s are identified as pentatricopeptide repeat (PPR) proteins, a large family of modular RNA-binding proteins that mediate several aspects of gene expression primarily in plant organelles. In this project we proposed to develop a system to test the ability of mtORFs in plants, which are closely related to known CMS factors. We will induce male fertility in various species of Brassicaceae, and test whether a down-relation in the expression of the recombinantCMS-genes restores fertility, using synthetically designed PPR proteins. 

2015 ◽  
Vol 71 (2) ◽  
pp. 196-208 ◽  
Author(s):  
Benjamin S. Gully ◽  
Kunal R. Shah ◽  
Mihwa Lee ◽  
Kate Shearston ◽  
Nicole M. Smith ◽  
...  

Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid α-hairpin motif. PPR proteins are typically single-stranded RNA-binding proteins with essential roles in organelle biogenesis, RNA editing and mRNA maturation. A modular, predictable code for sequence-specific binding of RNA by PPR proteins has recently been revealed, which opens the door to thede novodesign of bespoke proteins with specific RNA targets, with widespread biotechnological potential. Here, the design and production of a synthetic PPR protein based on a consensus sequence and the determination of its crystal structure to 2.2 Å resolution are described. The crystal structure displays helical disorder, resulting in electron density representing an infinite superhelical PPR protein. A structural comparison with related tetratricopeptide repeat (TPR) proteins, and with native PPR proteins, reveals key roles for conserved residues in directing the structure and function of PPR proteins. The designed proteins have high solubility and thermal stability, and can form long tracts of PPR repeats. Thus, consensus-sequence synthetic PPR proteins could provide a suitable backbone for the design of bespoke RNA-binding proteins with the potential for high specificity.


2021 ◽  
Vol 118 (50) ◽  
pp. e2109668118
Author(s):  
Ga Hye Kim ◽  
Ilmin Kwon

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins that control key events in RNA biogenesis under both normal and diseased cellular conditions. The low-complexity (LC) domain of hnRNPs can become liquid-like droplets or reversible amyloid-like polymers by phase separation. Yet, whether phase separation of the LC domains contributes to physiological functions of hnRNPs remains unclear. hnRNPH1 contains two LC domains, LC1 and LC2. Here, we show that reversible phase separation of the LC1 domain is critical for both interaction with different kinds of RNA-binding proteins and control of the alternative-splicing activity of hnRNPH1. Interestingly, although not required for phase separation, the LC2 domain contributes to the robust transcriptional activation of hnRNPH1 when fused to the DNA-binding domain, as found recently in acute lymphoblastic leukemia. Our data suggest that the ability of the LC1 domain to phase-separate into reversible polymers or liquid-like droplets is essential for function of hnRNPH1 as an alternative RNA-splicing regulator, whereas the LC2 domain may contribute to the aberrant transcriptional activity responsible for cancer transformation.


Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 591 ◽  
Author(s):  
Daria Mordovkina ◽  
Dmitry N. Lyabin ◽  
Egor A. Smolin ◽  
Ekaterina M. Sogorina ◽  
Lev P. Ovchinnikov ◽  
...  

Y-box binding proteins (YB proteins) are DNA/RNA-binding proteins belonging to a large family of proteins with the cold shock domain. Functionally, these proteins are known to be the most diverse, although the literature hardly offers any molecular mechanisms governing their activities in the cell, tissue, or the whole organism. This review describes the involvement of YB proteins in RNA-dependent processes, such as mRNA packaging into mRNPs, mRNA translation, and mRNA stabilization. In addition, recent data on the structural peculiarities of YB proteins underlying their interactions with nucleic acids are discussed.


2021 ◽  
Vol 15 ◽  
Author(s):  
Taro Ishiguro ◽  
Yoshitaka Nagai ◽  
Kinya Ishikawa

Spinocerebellar ataxia type 31 (SCA31) is a progressive neurodegenerative disease characterized by degeneration of Purkinje cells in the cerebellum. Its genetic cause is a 2.5- to 3.8-kb-long complex pentanucleotide repeat insertion containing (TGGAA)n, (TAGAA)n, (TAAAA)n, and (TAAAATAGAA)n located in an intron shared by two different genes: brain expressed associated with NEDD4-1 (BEAN1) and thymidine kinase 2 (TK2). Among these repeat sequences, (TGGAA)n repeat was the only sequence segregating with SCA31, which strongly suggests its pathogenicity. In SCA31 patient brains, the mutant BEAN1 transcript containing expanded UGGAA repeats (UGGAAexp) was found to form abnormal RNA structures called RNA foci in cerebellar Purkinje cell nuclei. In addition, the deposition of pentapeptide repeat (PPR) proteins, poly(Trp-Asn-Gly-Met-Glu), translated from UGGAAexp RNA, was detected in the cytoplasm of Purkinje cells. To uncover the pathogenesis of UGGAAexp in SCA31, we generated Drosophila models of SCA31 expressing UGGAAexp RNA. The toxicity of UGGAAexp depended on its length and expression level, which was accompanied by the accumulation of RNA foci and translation of repeat-associated PPR proteins in Drosophila, consistent with the observation in SCA31 patient brains. We also revealed that TDP-43, FUS, and hnRNPA2B1, motor neuron disease–linked RNA-binding proteins bound to UGGAAexp RNA, act as RNA chaperones to regulate the formation of RNA foci and repeat-associated translation. Further research on the role of RNA-binding proteins as RNA chaperones may also provide a novel therapeutic strategy for other microsatellite repeat expansion diseases besides SCA31.


2019 ◽  
Vol 60 (9) ◽  
pp. 1927-1938 ◽  
Author(s):  
Lauren K Dedow ◽  
Julia Bailey-Serres

Abstract Plants encode over 1800 RNA-binding proteins (RBPs) that modulate a myriad of steps in gene regulation from chromatin organization to translation, yet only a small number of these proteins and their target transcripts have been functionally characterized. Two classes of eukaryotic RBPs, pentatricopeptide repeat (PPR) and pumilio/fem-3 binding factors (PUF), recognize and bind to specific sequential RNA sequences through protein–RNA interactions. These modular proteins possess helical structural units containing key residues with high affinity for specific nucleotides, whose sequential order determines binding to a specific target RNA sequence. PPR proteins are nucleus-encoded, but largely regulate post-transcriptional gene regulation within plastids and mitochondria, including splicing, translation and RNA editing. Plant PUFs are involved in gene regulatory processes within the cell nucleus and cytoplasm. The modular structures of PPRs and PUFs that determine sequence specificity has facilitated identification of their RNA targets and biological functions. The protein-based RNA-targeting of PPRs and PUFs contrasts to the prokaryotic cluster regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) that target RNAs in prokaryotes. Together the PPR, PUF and CRISPR-Cas systems provide varied opportunities for RNA-targeted engineering applications.


2002 ◽  
Vol 99 (9) ◽  
pp. 5866-5871 ◽  
Author(s):  
M. Vermel ◽  
B. Guermann ◽  
L. Delage ◽  
J.-M. Grienenberger ◽  
L. Marechal-Drouard ◽  
...  

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