scholarly journals mRNP architecture in translating and stress conditions reveals an ordered pathway of mRNP compaction

2018 ◽  
Vol 217 (12) ◽  
pp. 4124-4140 ◽  
Author(s):  
Anthony Khong ◽  
Roy Parker
Keyword(s):  
Single Molecule ◽  
Mrna Stability ◽  
Rna Structure ◽  
Spatial Organization ◽  
Closed Loop ◽  
Protein Complexes ◽  
Loop Model ◽  
Reading Frame ◽  
Translation Inhibition ◽  
Changes Over Time

Stress granules (SGs) are transient membraneless organelles of nontranslating mRNA–protein complexes (mRNPs) that form during stress. In this study, we used multiple single-molecule FISH probes for particular mRNAs to examine their SG recruitment and spatial organization. Ribosome runoff is required for SG entry, as long open reading frame (ORF) mRNAs are delayed in SG accumulation, indicating that the SG transcriptome changes over time. Moreover, mRNAs are ∼20× compacted from an expected linear length when translating and compact ∼2-fold further in a stepwise manner beginning at the 5′ end during ribosome runoff. Surprisingly, the 5′ and 3′ ends of the examined mRNAs were separated when translating, but in nontranslating conditions the ends of long ORF mRNAs become close, suggesting that the closed-loop model of mRNPs preferentially forms on nontranslating mRNAs. Compaction of ribosome-free mRNAs is ATP independent, consistent with compaction occurring through RNA structure formation. These results suggest that translation inhibition triggers an mRNP reorganization that brings ends closer, which has implications for the regulation of mRNA stability and translation by 3′ UTR elements and the poly(A) tail.

scholarly journals mRNP architecture in translating and stress conditions reveals an ordered pathway of mRNP compaction

2018 ◽  
Author(s):  
Anthony Khong ◽  
Roy Parker
Keyword(s):  
Mrna Stability ◽  
Spatial Organization ◽  
Closed Loop ◽  
Stress Conditions ◽  
Loop Model ◽  
Translation Inhibition ◽  
Run Off ◽  
Specific Mrnas ◽  
Changes Over Time ◽  
Over Time

ABSTRACTStress granules (SGs) are non-translating mRNP assemblies that form during stress. Herein, we use multiple smFISH probes for specific mRNAs to examine their SG recruitment and spatial organization. We observed that ribosome run-off is required for SG entry with long ORF mRNAs being delayed in SG accumulation, revealing SG transcriptome changes over time. Moreover, mRNAs are ~20X compacted from an expected linear length when translating and compact ~2 fold further in a stepwise manner beginning at the 5’ end during ribosome run-off. Surprisingly, the 5’ and 3’ ends of the examined mRNAs were separated in non-stress conditions, but in non-translating conditions, the ends of AHNAK and DYNC1H1 mRNAs become close, suggesting the closed-loop model of mRNPs preferentially forms on non-translating mRNAs. These results suggest translation inhibition triggers a mRNP reorganization that brings ends closer, which has implications for the regulation of mRNA stability and translation by 3’ UTR elements and the poly(A) tail.

scholarly journals Spatial organization of single mRNPs at different stages of the gene expression pathway

2017 ◽  
Author(s):  
Srivathsan Adivarahan ◽  
Nathan Livingston ◽  
Beth Nicholson ◽  
Samir Rahman ◽  
Bin Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Cellular Localization ◽  
Spatial Organization ◽  
Closed Loop ◽  
Stress Granules ◽  
Mrna Metabolism ◽  
Ribonucleoprotein Complexes ◽  
Conformation Changes ◽  
Loop Conformation

AbstractmRNAs form ribonucleoprotein complexes (mRNPs) by association with proteins that are crucial for mRNA metabolism. While the mRNP proteome has been well characterized, little is known about mRNP organization. Using a single molecule approach, we show that mRNA conformation changes depending on its cellular localization and translational state. Compared to nuclear mRNPs and lncRNPs, association with ribosomes decompacts individual mRNAs, while their sequestration into stress-granules leads to increased compaction. Moreover, translating mRNAs rarely show co-localizing 5’ and 3’ ends, indicating that mRNAs are either not translated in a closed-loop configuration, or that mRNA circularization is transient, suggesting that a stable closed-loop conformation is not a universal state for all translating mRNAs.One Sentence SummarySingle mRNA studies in cells show RNA compaction changes depending on translational state, but mRNAs are not translated in closed-loop conformation.

scholarly journals Destabilization of Eukaryote mRNAs by 5′ Proximal Stop Codons Can Occur Independently of the Nonsense-Mediated mRNA Decay Pathway

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 800 ◽  
Author(s):  
Barbara Gorgoni ◽  
Yun-Bo Zhao ◽  
J. Krishnan ◽  
Ian Stansfield
Keyword(s):  
Mrna Stability ◽  
Mrna Decay ◽  
Closed Loop ◽  
Stop Codon ◽  
Translation Termination ◽  
Translation System ◽  
Translation Elongation ◽  
Reading Frame ◽  
Stop Codons ◽  
Nonsense Mediated Mrna Decay

In eukaryotes, the binding of poly(A) binding protein (PAB) to the poly(A) tail is central to maintaining mRNA stability. PABP interacts with the translation termination apparatus, and with eIF4G to maintain 3′–5′ mRNA interactions as part of an mRNA closed loop. It is however unclear how ribosome recycling on a closed loop mRNA is influenced by the proximity of the stop codon to the poly(A) tail, and how post-termination ribosome recycling affects mRNA stability. We show that in a yeast disabled for nonsense mediated mRNA decay (NMD), a PGK1 mRNA with an early stop codon at codon 22 of the reading frame is still highly unstable, and that this instability cannot be significantly countered even when 50% stop codon readthrough is triggered. In an NMD-deficient mutant yeast, stable reporter alleles with more 3′ proximal stop codons could not be rendered unstable through Rli1-depletion, inferring defective Rli1 ribosome recycling is insufficient in itself to trigger mRNA instability. Mathematical modelling of a translation system including the effect of ribosome recycling and poly(A) tail shortening supports the hypothesis that impaired ribosome recycling from 5′ proximal stop codons may compromise initiation processes and thus destabilize the mRNA. A model is proposed wherein ribosomes undergo a maturation process during early elongation steps, and acquire competency to re-initiate on the same mRNA as translation elongation progresses beyond the very 5′ proximal regions of the mRNA.

scholarly journals Fast Diffusion of the Unassembled PetC1-GFP Protein in the Cyanobacterial Thylakoid Membrane

Life ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 15
Author(s):  
Radek Kaňa ◽  
Gábor Steinbach ◽  
Roman Sobotka ◽  
György Vámosi ◽  
Josef Komenda
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Protein Interactions ◽  
Thylakoid Membrane ◽  
Protein Complexes ◽  
Correlation Spectroscopy ◽  
Membrane Microdomains ◽  
Fast Diffusion ◽  
Light Harvesting Complexes ◽  
Term Survival

Biological membranes were originally described as a fluid mosaic with uniform distribution of proteins and lipids. Later, heterogeneous membrane areas were found in many membrane systems including cyanobacterial thylakoids. In fact, cyanobacterial pigment–protein complexes (photosystems, phycobilisomes) form a heterogeneous mosaic of thylakoid membrane microdomains (MDs) restricting protein mobility. The trafficking of membrane proteins is one of the key factors for long-term survival under stress conditions, for instance during exposure to photoinhibitory light conditions. However, the mobility of unbound ‘free’ proteins in thylakoid membrane is poorly characterized. In this work, we assessed the maximal diffusional ability of a small, unbound thylakoid membrane protein by semi-single molecule FCS (fluorescence correlation spectroscopy) method in the cyanobacterium Synechocystis sp. PCC6803. We utilized a GFP-tagged variant of the cytochrome b6f subunit PetC1 (PetC1-GFP), which was not assembled in the b6f complex due to the presence of the tag. Subsequent FCS measurements have identified a very fast diffusion of the PetC1-GFP protein in the thylakoid membrane (D = 0.14 − 2.95 µm2s−1). This means that the mobility of PetC1-GFP was comparable with that of free lipids and was 50–500 times higher in comparison to the mobility of proteins (e.g., IsiA, LHCII—light-harvesting complexes of PSII) naturally associated with larger thylakoid membrane complexes like photosystems. Our results thus demonstrate the ability of free thylakoid-membrane proteins to move very fast, revealing the crucial role of protein–protein interactions in the mobility restrictions for large thylakoid protein complexes.

scholarly journals Probing the biogenesis pathway and dynamics of thylakoid membranes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tuomas Huokko ◽  
Tao Ni ◽  
Gregory F. Dykes ◽  
Deborah M. Simpson ◽  
Philip Brownridge ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Photosystem I ◽  
Spatial Organization ◽  
Electron Flux ◽  
Protein Complexes ◽  
Thylakoid Membranes ◽  
Thylakoid Biogenesis ◽  
Photosynthetic Complexes ◽  
Photosynthetic Machinery ◽  
Pcc 7942

AbstractHow thylakoid membranes are generated to form a metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery.

scholarly journals Translational and Structural Requirements of the Early Nodulin Gene enod40, a Short-Open Reading Frame-Containing RNA, for Elicitation of a Cell-Specific Growth Response in the Alfalfa Root Cortex

2001 ◽  
Vol 21 (1) ◽  
pp. 354-366 ◽  
Author(s):  
Carolina Sousa ◽  
Christina Johansson ◽  
Celine Charon ◽  
Hamid Manyani ◽  
Christof Sautter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Rna Structure ◽  
Open Reading Frames ◽  
In Vitro Translation ◽  
Cortical Cells ◽  
Root Cortex ◽  
Reading Frame ◽  
Early Nodulin

ABSTRACT A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin geneenod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5′ and 3′ regions ofenod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

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