-
Biomolecular NMR Assignments Jun 2024Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA....
Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA's depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets.
Topics: Ricin; Nuclear Magnetic Resonance, Biomolecular; Nitrogen Isotopes; Protein Subunits; Amino Acid Sequence
PubMed: 38642265
DOI: 10.1007/s12104-024-10172-8 -
Nature Communications Apr 2024DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions...
DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions are currently unknown. In this study, we present cryo-EM reconstructions of nucleolar pre-60S intermediates that reveal an unexpected, alternate secondary structure within the nascent peptidyl-transferase-center (PTC). Our analysis of three sequential nucleolar pre-60S intermediates reveals that the DEAD-box ATPase Dbp10/DDX54 remodels this alternate base pairing and enables the formation of the rRNA junction that anchors the mature form of the universally conserved PTC A-loop. Post-catalysis, Dbp10 captures rRNA helix H61, initiating the concerted exchange of biogenesis factors during late nucleolar 60S maturation. Our findings show that Dbp10 activity is essential for the formation of the ribosome active site and reveal how this function is integrated with subsequent assembly steps to drive the biogenesis of the large ribosomal subunit.
Topics: DEAD-box RNA Helicases; Peptidyl Transferases; Ribosomal Proteins; Ribosome Subunits, Large, Eukaryotic; Ribosomes; RNA, Ribosomal; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 38632236
DOI: 10.1038/s41467-024-47616-7 -
Cell Reports May 2024Scanning and initiation are critical steps in translation. Here, we utilized translation complex profiling (TCP-seq) to investigate 48S organization and eIF4G1-eIF1...
Scanning and initiation are critical steps in translation. Here, we utilized translation complex profiling (TCP-seq) to investigate 48S organization and eIF4G1-eIF1 inhibition impact. We provide global views of scanning and leaky scanning, uncovering a central role of eIF4G1-eIF1 in their regulation. We confirm AUG context importance, with non-leaky genes featuring a Kozak context and cytosine at positions -1 and +5. Capturing 48S complexes associated with eIF1, eIF4G1, eIF3, and eIF2 through selective TCP-seq revealed that the eIF3-scanning ribosome is highly vulnerable to eIF4G1-eIF1 inhibition, and eIF1 tends to dissociate upon AUG recognition. Initiation-site footprint analysis revealed a class spanning -12 to +18/19 from the AUG, representing the entire 48S and enriched with eIF2, eIF1, and eIF4G1, indicative of early initiation. Another eIF3-dependent class extends up to +26 and exhibits reduced eIF2 and eIF4G1 association, suggesting a late/alternative initiation complex. Our analysis provides an overview of scanning, initiation, and evidence for conformational rearrangements in vivo.
Topics: Ribosomes; Saccharomyces cerevisiae; Peptide Chain Initiation, Translational; Humans; Eukaryotic Initiation Factor-4G; Protein Biosynthesis; Saccharomyces cerevisiae Proteins
PubMed: 38630588
DOI: 10.1016/j.celrep.2024.114126 -
ELife Apr 2024GlcNAcylation is a dynamic post-translational modification that diversifies the proteome. Its dysregulation is associated with neurological disorders that impair...
GlcNAcylation is a dynamic post-translational modification that diversifies the proteome. Its dysregulation is associated with neurological disorders that impair cognitive function, and yet identification of phenotype-relevant candidate substrates in a brain-region specific manner remains unfeasible. By combining an GlcNAc binding activity derived from OGA (OGA) with TurboID proximity labeling in , we developed an GlcNAcylation profiling tool that translates GlcNAc modification into biotin conjugation for tissue-specific candidate substrates enrichment. We mapped the GlcNAc interactome in major brain regions of and found that components of the translational machinery, particularly ribosomal subunits, were abundantly GlcNAcylated in the mushroom body of brain. Hypo-GlcNAcylation induced by ectopic expression of active OGA in the mushroom body decreased local translational activity, leading to olfactory learning deficits that could be rescued by dMyc overexpression-induced increase of protein synthesis. Our study provides a useful tool for future dissection of tissue-specific functions of GlcNAcylation in , and suggests a possibility that GlcNAcylation impacts cognitive function via regulating regional translational activity in the brain.
Topics: Animals; Drosophila; Mushroom Bodies; Brain; Cognition; Protein Processing, Post-Translational
PubMed: 38619103
DOI: 10.7554/eLife.91269 -
Nature Communications Apr 2024In chloroplasts, insertion of proteins with multiple transmembrane domains (TMDs) into thylakoid membranes usually occurs in a co-translational manner. Here, we have...
In chloroplasts, insertion of proteins with multiple transmembrane domains (TMDs) into thylakoid membranes usually occurs in a co-translational manner. Here, we have characterized a thylakoid protein designated FPB1 (Facilitator of PsbB biogenesis1) which together with a previously reported factor PAM68 (Photosynthesis Affected Mutant68) is involved in assisting the biogenesis of CP47, a subunit of the Photosystem II (PSII) core. Analysis by ribosome profiling reveals increased ribosome stalling when the last TMD segment of CP47 emerges from the ribosomal tunnel in fpb1 and pam68. FPB1 interacts with PAM68 and both proteins coimmunoprecipitate with SecY/E and Alb3 as well as with some ribosomal components. Thus, our data indicate that, in coordination with the SecY/E translocon and the Alb3 integrase, FPB1 synergistically cooperates with PAM68 to facilitate the co-translational integration of the last two CP47 TMDs and the large loop between them into thylakoids and the PSII core complex.
Topics: Chloroplasts; Photosystem II Protein Complex; Ribosomes; Thylakoids
PubMed: 38600073
DOI: 10.1038/s41467-024-46863-y -
Life Science Alliance Jun 2024Translational regulation by non-coding RNAs is a mechanism commonly used by cells to fine-tune gene expression. A fragment derived from an archaeal valine tRNA (Val-tRF)...
Translational regulation by non-coding RNAs is a mechanism commonly used by cells to fine-tune gene expression. A fragment derived from an archaeal valine tRNA (Val-tRF) has been previously identified to bind the small subunit of the ribosome and inhibit translation in Here, we present three cryo-electron microscopy structures of Val-tRF bound to the small subunit of ribosomes at resolutions between 4.02 and 4.53 Å. Within these complexes, Val-tRF was observed to bind to conserved RNA-interacting sites, including the ribosomal decoding center. The binding of Val-tRF destabilizes helices h24, h44, and h45 and the anti-Shine-Dalgarno sequence of 16S rRNA. The binding position of this molecule partially overlaps with the translation initiation factor aIF1A and occludes the mRNA P-site codon. Moreover, we found that the binding of Val-tRF is associated with steric hindrance of the H69 base of 23S rRNA in the large ribosome subunit, thereby preventing 70S assembly. Our data exemplify how tRNA-derived fragments bind to ribosomes and provide new insights into the mechanisms underlying translation inhibition by Val-tRFs.
Topics: RNA, Ribosomal, 16S; Cryoelectron Microscopy; Ribosomes; RNA, Transfer; Valine
PubMed: 38599770
DOI: 10.26508/lsa.202302488 -
Experimental and Therapeutic Medicine May 2024Essential tremor (ET) and Parkinson's disease (PD) are common chronic movement disorders that can cause a substantial degree of disability. However, the etiology...
Essential tremor (ET) and Parkinson's disease (PD) are common chronic movement disorders that can cause a substantial degree of disability. However, the etiology underlying these two conditions remains poorly understood. In the present study, Whole-exome sequencing of peripheral blood samples from the proband and Sanger sequencing of the other 18 family members, and pedigree analysis of four generations of 29 individuals with both ET and PD in a nonconsanguineous Chinese family were performed. Specifically, family members who had available medical information, including historical documentation and physical examination records, were included. A novel c.1909A>T (p.Ser637Cys) missense mutation was identified in the eukaryotic translation initiation factor 4γ1 () gene as the candidate likely responsible for both conditions. In total, 9 family members exhibited tremor of the bilateral upper limbs and/or head starting from ages of ≥40 years, 3 of whom began showing evidence of PD in their 70s. Eukaryotic initiation factor 4 (eIF4)G1, a component of the translation initiation complex eIF4F, serves as a scaffold protein that interacts with many initiation factors and then binds to the 40S ribosomal subunit. The (p.Ser637Cys) might inhibit the recruitment of the mRNA to the ribosome. In conclusion, the results from the present study suggested that may be responsible for the hereditary PD with 'antecedent ET' reported in the family assessed.
PubMed: 38590578
DOI: 10.3892/etm.2024.12494 -
RNA (New York, N.Y.) Jun 2024Ribosomes translate mRNA into proteins and are essential for every living organism. In eukaryotes, both ribosomal subunits are rapidly assembled in a strict hierarchical...
Ribosomes translate mRNA into proteins and are essential for every living organism. In eukaryotes, both ribosomal subunits are rapidly assembled in a strict hierarchical order, starting in the nucleolus with the transcription of a common precursor ribosomal RNA (pre-rRNA). This pre-rRNA encodes three of the four mature rRNAs, which are formed by several, consecutive endonucleolytic and exonucleolytic processing steps. Historically, northern blots are used to analyze the variety of different pre-rRNA species, only allowing rough length estimations. Although this limitation can be overcome with primer extension, both approaches often use radioactivity and are time-consuming and costly. Here, we present "Riboprobing," a linker ligation-based workflow followed by reverse transcription and PCR for easy and fast detection and characterization of pre-rRNA species and their 5' as well as 3' ends. Using standard molecular biology laboratory equipment, "Riboprobing" allows reliable discrimination of pre-rRNA species not resolved by northern blot (e.g., 27SA, 27SA, and 27SB pre-rRNA). The method can successfully be used for the analysis of total cell extracts as well as purified pre-ribosomes for a straightforward evaluation of the impact of mutant gene versions or inhibitors. In the course of method development, we identified and characterized a hitherto undescribed aberrant pre-rRNA arising from LiCl inhibition. This pre-rRNA fragment spans from processing site A1 to E, forming a small RNP that lacks most early joining assembly factors. This finding expands our knowledge of how the cell deals with severe pre-rRNA processing defects and demonstrates the strict requirement for the 5'ETS (external transcribed spacer) for the assembly process.
Topics: RNA Precursors; RNA, Ribosomal; Workflow; RNA Processing, Post-Transcriptional
PubMed: 38580456
DOI: 10.1261/rna.079912.123 -
Frontiers in Microbiology 2024Porcine epidemic diarrhea (PED) is an acute, highly contagious, and high-mortality enterophilic infectious disease caused by the porcine epidemic diarrhea virus (PEDV)....
INTRODUCTION
Porcine epidemic diarrhea (PED) is an acute, highly contagious, and high-mortality enterophilic infectious disease caused by the porcine epidemic diarrhea virus (PEDV). PEDV is globally endemic and causes substantial economic losses in the swine industry. The PEDV E protein is the smallest structural protein with high expression levels that interacts with the M protein and participates in virus assembly. However, how the host proteins interact with E proteins in PEDV replication remains unknown.
METHODS
We identified host proteins that interact with the PEDV E protein using a combination of PEDV E protein-labeled antibody co-immunoprecipitation and tandem liquid-chromatography mass-spectroscopy (LC-MS/MS).
RESULTS
Bioinformatical analysis showed that in eukaryotes, ribosome biogenesis, RNA transport, and amino acid biosynthesis represent the three main pathways that are associated with the E protein. The interaction between the E protein and isocitrate dehydrogenase [NAD] β-subunit (NAD-IDH-β), DNA-directed RNA polymerase II subunit RPB9, and mRNA-associated protein MRNP 41 was validated using co-immunoprecipitation and confocal assays. NAD-IDH-β overexpression significantly inhibited viral replication.
DISCUSSION
The antiviral effect of NAD-IDH-β suggesting that the E protein may regulate host metabolism by interacting with NAD-IDH-β, thereby reducing the available energy for viral replication. Elucidating the interaction between the PEDV E protein and host proteins may clarify its role in viral replication. These results provide a theoretical basis for the study of PEDV infection mechanism and antiviral targets.
PubMed: 38577683
DOI: 10.3389/fmicb.2024.1380578 -
RNA (New York, N.Y.) Jun 202430S subunits become inactive upon exposure to low Mg concentration, because of a reversible conformational change that entails nucleotides (nt) in the neck helix (h28)...
30S subunits become inactive upon exposure to low Mg concentration, because of a reversible conformational change that entails nucleotides (nt) in the neck helix (h28) and 3' tail of 16S rRNA. This active-to-inactive transition involves partial unwinding of h28 and repairing of nt 921-923 with nt 1532-1534, which requires flipping of the 3' tail by ∼180°. Growing evidence suggests that immature 30S particles adopt the inactive conformation in the cell, and transition to the active state occurs at a late stage of maturation. Here, we target nucleotides that form the alternative helix (hALT) of the inactive state. Using an orthogonal ribosome system, we find that disruption of hALT decreases translation activity in the cell modestly, by approximately twofold, without compromising ribosome fidelity. Ribosomes carrying substitutions at positions 1532-1533 support the growth of strain Δ7 prrn (which carries a single rRNA operon), albeit at rates 10%-20% slower than wild-type ribosomes. These mutant Δ7 prrn strains accumulate free 30S particles and precursor 17S rRNA, indicative of biogenesis defects. Analysis of purified control and mutant subunits suggests that hALT stabilizes the inactive state by 1.2 kcal/mol with little-to-no impact on the active state or the transition state of conversion.
Topics: RNA, Ribosomal, 16S; Nucleic Acid Conformation; Escherichia coli; Ribosome Subunits, Small, Bacterial; Protein Biosynthesis; Magnesium
PubMed: 38570183
DOI: 10.1261/rna.079960.124