Mechanistic Insight into Pentatricopeptide Repeat Proteins as Sequence-Specific RNA-Binding Proteins for Organellar RNAs in Plants

Pentatricopeptide repeat (PPR) proteins are RNA binding proteins with functions in organelle RNA metabolism. They are found in all eukaryotes but have been most extensively studied in plants. We report on the identification of 12 PPR-encoding genes in the genome of the protistDictyostelium discoideum, with potential homologs in other members of the same lineage and some predicted novel functions for the encoded gene products in protists. For one of the gene products, we show that it localizes to the mitochondria, and we also demonstrate that antisense inhibition of its expression leads to slower growth, a phenotype associated with mitochondrial dysfunction.

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In vivo stabilization of endogenous chloroplast RNAs by customized artificial pentatricopeptide repeat proteins

10.1101/2020.11.21.392746 ◽

2020 ◽

Author(s):

Nikolay Manavski ◽

Louis-Valentin Meteignier ◽

Margarita Rojas ◽

Andreas Brachmann ◽

Alice Barkan ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Sequence Specificity ◽

Pentatricopeptide Repeat ◽

Functional Capabilities ◽

Ppr Protein ◽

Repeat Proteins ◽

Ppr Proteins ◽

Physical Barriers

ABSTRACTPentatricopeptide repeat (PPR) proteins are helical repeat-proteins that bind RNA in a modular fashion with a sequence-specificity that can be manipulated by the use of an amino acid code. As such, PPR repeats are promising scaffolds for the design of RNA binding proteins for synthetic biology applications. However, the in vivo functional capabilities of artificial PPR proteins built from consensus PPR motifs are just starting to be explored. Here, we report in vivo functions of an artificial PPR protein, dPPRrbcL, made of consensus PPR motifs that were designed to bind a sequence near the 5’ end of rbcL transcripts in Arabidopsis chloroplasts. We used a functional complementation assay to demonstrate that this protein bound its intended RNA target with specificity in vivo and that it substituted for a natural PPR protein by stabilizing processed rbcL mRNA. We targeted a second protein of analogous design to the petL 5’ UTR, where it substituted for the native stabilizing PPR protein PGR3, albeit inefficiently. These results showed that artificial PPRs can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases.

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RNA-binding specificity landscapes of designer pentatricopeptide repeat proteins elucidate principles of PPR–RNA interactions

Nucleic Acids Research ◽

10.1093/nar/gkx1288 ◽

2017 ◽

Vol 46 (5) ◽

pp. 2613-2623 ◽

Cited By ~ 16

Author(s):

Rafael G Miranda ◽

James J McDermott ◽

Alice Barkan

Keyword(s):

Rna Binding ◽

Binding Specificity ◽

Pentatricopeptide Repeat ◽

Repeat Proteins ◽

Pentatricopeptide Repeat Proteins

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Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants

The Plant Journal ◽

10.1111/tpj.13121 ◽

2016 ◽

Vol 85 (4) ◽

pp. 532-547 ◽

Cited By ~ 142

Author(s):

Shifeng Cheng ◽

Bernard Gutmann ◽

Xiao Zhong ◽

Yongtao Ye ◽

Mark F. Fisher ◽

...

Keyword(s):

Rna Editing ◽

Rna Binding ◽

Land Plants ◽

Pentatricopeptide Repeat ◽

Structural Motifs ◽

Repeat Proteins ◽

Pentatricopeptide Repeat Proteins

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Identification of clustered organellar short (cos) RNAs and of a conserved family of organellar RNA-binding proteins, the heptatricopeptide repeat proteins, in the malaria parasite

Nucleic Acids Research ◽

10.1093/nar/gky710 ◽

2018 ◽

Cited By ~ 2

Author(s):

Arne Hillebrand ◽

Joachim M Matz ◽

Martin Almendinger ◽

Katja Müller ◽

Kai Matuschewski ◽

...

Keyword(s):

Malaria Parasite ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Repeat Proteins

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The design and structural characterization of a synthetic pentatricopeptide repeat protein

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714024869 ◽

2015 ◽

Vol 71 (2) ◽

pp. 196-208 ◽

Cited By ~ 23

Author(s):

Benjamin S. Gully ◽

Kunal R. Shah ◽

Mihwa Lee ◽

Kate Shearston ◽

Nicole M. Smith ◽

...

Keyword(s):

Crystal Structure ◽

Binding Proteins ◽

Rna Binding ◽

De Novo ◽

Rna Binding Proteins ◽

Specific Binding ◽

Consensus Sequence ◽

Pentatricopeptide Repeat ◽

Ppr Protein ◽

Ppr Proteins

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.

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RNA-binding Proteins in Neurospora crassa: An Insight into Spliceosome and Post Transcriptional Gene Silencing Machinery

Current Proteomics ◽

10.2174/1570164614666170329145814 ◽

2017 ◽

Vol 14 (4) ◽

Author(s):

Kunal Zaveri ◽

Patnala Kiranmayi ◽

M. Nagamruta ◽

K. Vijaya Rachel

Keyword(s):

Gene Silencing ◽

Neurospora Crassa ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Transcriptional Gene Silencing ◽

Post Transcriptional Gene Silencing ◽

Insight Into

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Combinatorial Genetics of RNA Binding Proteins for Lifespan Regulation in C. elegans

10.25172/jour.4.1.6 ◽

2019 ◽

Vol 4 (Spring 2019) ◽

Author(s):

Nikita Tapiawala

Keyword(s):

Gene Regulation ◽

Aging Process ◽

Transcriptional Control ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

C Elegans ◽

Experimental Laboratory ◽

Control Post ◽

Insight Into

RNA binding proteins control post-transcriptional aspects of gene regulation. While transcriptional control over the aging process has been well-established, less is known about post-transcriptional control over the aging process. We have used CRISPR/Cas9 genetics to study combinations of RNA binding proteins in C. elegan, a transparent nematode or roundworm one millimeter in length, and their role in aging. The manner in which the research was conducted was through experimental laboratory work in which the lifespan of the nematodes was monitored until their death. The three mutations affecting RNA binding proteins that assays were performed with are exc-7, fox-1, and mbl-1. We found that double mutants have stronger effects on lifespan than single mutants. Thus, RNA binding protein mutations, although separate, appeared to be functionally connected. These observations, though being considered in C. elegans, could have a substantive impact on the study of gene regulation in humans and in turn, provide insight into the human aging processes.

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New Insight into Cold Shock Proteins: RNA-binding Proteins Involved in Stress Response and Virulence

Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria ◽

10.1002/9781119004813.ch85 ◽

2016 ◽

pp. 873-880

Author(s):

Charlotte Michaux ◽

Jean-Christophe Giard

Keyword(s):

Stress Response ◽

Binding Proteins ◽

Cold Shock ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cold Shock Proteins ◽

Shock Proteins ◽

Insight Into

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Searching for a Match: Structure, Function and Application of Sequence-Specific RNA-Binding Proteins

Plant and Cell Physiology ◽

10.1093/pcp/pcz072 ◽

2019 ◽

Vol 60 (9) ◽

pp. 1927-1938 ◽

Cited By ~ 6

Author(s):

Lauren K Dedow ◽

Julia Bailey-Serres

Keyword(s):

Gene Regulation ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Sequence Specificity ◽

Pentatricopeptide Repeat ◽

Rna Sequences ◽

Regulatory Processes ◽

Ppr Proteins ◽

Modular Structures

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.

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