bims-tricox 2022-08-21 papers

bims-tricox

Biomed News

on Translation, ribosomes and COX

Issue of 2022–08–21
ten papers selected by
Yash Verma, University of Delhi South Campus

In vitro characterization of Dhr1 from Saccharomyces cerevisiae.
Attacking a DEAD problem: The role of DEAD-box ATPases in ribosome assembly and beyond.
Lysate and cell-based assays to probe the translational role of RNA helicases.
A Conundrum of r-Protein Stability: Unbalanced Stoichiometry of r-Proteins during Stationary Phase in Escherichia coli.
Mitochondrial microproteins link metabolic cues to respiratory chain biogenesis.
Evaluating data integrity in ribosome footprinting datasets through modelled polysome profiles.
Mitochondrial mRNA localization is governed by translation kinetics and spatial transport.
Human eukaryotic initiation factor 4E (eIF4E) and the nucleotide-bound state of eIF4A regulate eIF4F binding to RNA.
Evidence for low-level translation in human erythrocytes.
How much virus does a person with COVID exhale? New research has answers.

Methods Enzymol. 2022 ;pii: S0076-6879(22)00149-5. [Epub ahead of print]673 77-101

In vitro characterization of Dhr1 from Saccharomyces cerevisiae.

Ran Lin, Carl C Correll, Arlen W Johnson.

  The RNA helicase Dhr1 from S. cerevisiae is an essential enzyme required for the assembly of the cytosolic small ribosomal subunit (SSU). A critical feature of the SSU is the central pseudoknot, an RNA fold that organizes the overall architecture of the subunit and connects all four domains of the 18S ribosomal RNA (rRNA). The initial folding of rRNA is guided, in part, by the U3 small nucleolar RNA, which base-pairs with the pre-rRNA in such a way as to preclude premature formation of the central pseudoknot. Thus, the essential role of Dhr1 is the unwinding of U3 from the pre-rRNA to allow folding of the central pseudoknot. Enzymes of the DEAH/RNA helicase A-like (RHA) family, to which Dhr1 belongs, are involved in splicing and ribosome biogenesis. They typically unwind RNA duplexes by translocation along a single strand of RNA in a 3' to 5' direction, driven by ATP hydrolysis. The substrate specificity of these enzymes requires tight regulation of their activity, by restricting access to their substrates, requiring adaptors to recruit them to their substrates and mechanisms of inhibiting and activating their activity. Purified Dhr1 is an active RNA-dependent ATPase with specific unwinding activity. Here, we provide detailed protocols for its purification and assays for its ATPase and unwinding activities.

Keywords:  DEAH/RHA helicase; DHR1; RIBOSOME; RIBOSOME ASSEMBLY; SSU processome; U3 snoRNA

DOI:  https://doi.org/10.1016/bs.mie.2022.03.058
Methods Enzymol. 2022 ;pii: S0076-6879(22)00124-0. [Epub ahead of print]673 19-38

Attacking a DEAD problem: The role of DEAD-box ATPases in ribosome assembly and beyond.

Katrin Karbstein.

  DEAD-box proteins are a subfamily of ATPases with similarity to RecA-type helicases that are involved in all aspects of RNA Biology. Despite their potential to regulate these processes via their RNA-dependent ATPase activity, their roles remain poorly characterized. Here I describe a roadmap to study these proteins in the context of ribosome assembly, the process that utilizes more than half of all DEAD-box proteins encoded in the yeast genome.

Keywords:  DEAD box protein roles; Dbp5; Dbp7; Has1; Prp5; Prpr28; Rok1; Sub2

DOI:  https://doi.org/10.1016/bs.mie.2022.03.033
Methods Enzymol. 2022 ;pii: S0076-6879(22)00123-9. [Epub ahead of print]673 141-168

Lysate and cell-based assays to probe the translational role of RNA helicases.

Kevin C Wilkins, Srivats Venkataramanan, Stephen N Floor.

  Translation initiation is the first step in protein synthesis, during which the small subunit of the ribosome scans the 5' untranslated region (5'UTR) of an mRNA to identify a start codon and commence translation elongation. By unwinding and modulating secondary structures and other RNA features present in the 5'UTR, RNA helicases can regulate ribosome scanning and start codon selection. This chapter presents an approach to measure the effect of RNA helicases on mRNA translation initiation. 5'UTR luciferase reporters are transcribed in vitro and employed in either of two assays. The in vitro assay translates the reporters in a cell-free whole-cell lysate system, which allows for greater biochemical manipulation and tighter control over confounding effects. In the alternative cell-based approach, the reporters are transfected and translated in living cells, which provides a more physiological setup. Either method can be used to investigate how the perturbation of a helicase, such as changes in protein levels or mutations, affects translation initiation at the 5'UTR level. The chapter also discusses alternative approaches, troubleshooting, and further applications of these methods. These assays will provide insights on the role of helicases and other translational factors as regulators of the proteome both in physiological and diseased settings.

Keywords:  DEAD-box proteins; Luciferase assay; Protein synthesis; RNA chaperones; Translational control

DOI:  https://doi.org/10.1016/bs.mie.2022.03.032
mBio. 2022 Aug 18. e0187322

A Conundrum of r-Protein Stability: Unbalanced Stoichiometry of r-Proteins during Stationary Phase in Escherichia coli.

Kaspar Reier, Petri-Jaan Lahtvee, Aivar Liiv, Jaanus Remme.

  Bacterial ribosomes are composed of three rRNA and over 50 ribosomal protein (r-protein) molecules. r-proteins are essential for ribosome assembly and structural stability and also participate in almost all ribosome functions. Ribosomal components are present in stoichiometric amounts in the mature 70S ribosomes during exponential and early stationary growth phases. Ribosomes are degraded in stationary phase; however, the stability and fate of r-proteins during stationary growth phase are not known. In this study, we report a quantitative analysis of ribosomal components during extended stationary-phase growth in Escherichia coli. We show that (i) the quantity of ribosomes per cell mass decreases in stationary phase, (ii) 70S ribosomes contain r-proteins in stoichiometric amounts, (iii) 30S subunits are degraded faster than 50S subunits, (iv) the quantities of 21 r-proteins in the total proteome decrease during 14 days (short-lived r-proteins) concomitantly with the reduction of cellular RNA, and (e) 30 r-proteins are stable and form a pool of free r-proteins (stable r-proteins). Thus, r-proteins are present in nonstoichiometric amounts in the proteome of E. coli during the extended stationary phase. IMPORTANCE Ribosome degradation has been extensively described from the viewpoint of its main component, rRNA. Here, we aim to complement our knowledge by quantitatively analyzing r-protein degradation and stability both in the ribosomes and in the whole-cell proteome during stationary phase in E. coli. r-proteins are considered to be very stable in the proteome. Here, we show that a specific set of r-proteins are rapidly degraded after release from the rRNA. The degradation of r-proteins is an intriguing new aspect of r-protein metabolism in bacteria.

Keywords:  proteomics; rRNA; ribosomal proteins; ribosomes; stationary phase

DOI:  https://doi.org/10.1128/mbio.01873-22
Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01021-X. [Epub ahead of print]40(7): 111204

Mitochondrial microproteins link metabolic cues to respiratory chain biogenesis.

Chao Liang, Shan Zhang, David Robinson, Matthew Vander Ploeg, Rebecca Wilson, Jiemin Nah, Dale Taylor, Sheryl Beh, Radiance Lim, Lei Sun, Deborah M Muoio, David A Stroud, Lena Ho.

  Electron transport chain (ETC) biogenesis is tightly coupled to energy levels and availability of ETC subunits. Complex III (CIII), controlling ubiquinol:ubiquinone ratio in ETC, is an attractive node for modulating ETC levels during metabolic stress. Here, we report the discovery of mammalian Co-ordinator of mitochondrial CYTB (COM) complexes that regulate the stepwise CIII biogenesis in response to nutrient and nuclear-encoded ETC subunit availability. The COMA complex, consisting of UQCC1/2 and membrane anchor C16ORF91, facilitates translation of CIII enzymatic core subunit CYTB. Subsequently, microproteins SMIM4 and BRAWNIN together with COMA subunits form the COMB complex to stabilize nascent CYTB. Finally, UQCC3-containing COMC facilitates CYTB hemylation and association with downstream CIII subunits. Furthermore, when nuclear CIII subunits are limiting, COMB is required to chaperone nascent CYTB to prevent OXPHOS collapse. Our studies highlight CYTB synthesis as a key regulatory node of ETC biogenesis and uncover the roles of microproteins in maintaining mitochondrial homeostasis.

Keywords:  CP: Metabolism; CYTB; SEPs; SMIM4; UQCC1; UQCC2; complex III; electron transport chain; microproteins; nuclear-mitochondrial coordination; smORFs

DOI:  https://doi.org/10.1016/j.celrep.2022.111204
Nucleic Acids Res. 2022 Aug 18. pii: gkac705. [Epub ahead of print]

Evaluating data integrity in ribosome footprinting datasets through modelled polysome profiles.

Fabio Hedayioglu, Emma J Mead, Patrick B F O'Connor, Matas Skiotys, Owen J Sansom, Giovanna R Mallucci, Anne E Willis, Pavel V Baranov, C Mark Smales, Tobias von der Haar.

The assessment of transcriptome-wide ribosome binding to mRNAs is useful for studying the dynamic regulation of protein synthesis. Two methods frequently applied in eukaryotic cells that operate at different levels of resolution are polysome profiling, which reveals the distribution of ribosome loads across the transcriptome, and ribosome footprinting (also termed ribosome profiling or Ribo-Seq), which when combined with appropriate data on mRNA expression can reveal ribosome densities on individual transcripts. In this study we develop methods for relating the information content of these two methods to one another, by reconstructing theoretical polysome profiles from ribosome footprinting data. Our results validate both approaches as experimental tools. Although we show that both methods can yield highly consistent data, some published ribosome footprinting datasets give rise to reconstructed polysome profiles with non-physiological features. We trace these aberrant features to inconsistencies in RNA and Ribo-Seq data when compared to datasets yielding physiological polysome profiles, thereby demonstrating that modelled polysomes are useful for assessing global dataset properties such as its quality in a simple, visual approach. Aside from using polysome profile reconstructions on published datasets, we propose that this also provides a useful tool for validating new ribosome footprinting datasets in early stages of analyses.

DOI: https://doi.org/10.1093/nar/gkac705
PLoS Comput Biol. 2022 Aug 19. 18(8): e1010413

Mitochondrial mRNA localization is governed by translation kinetics and spatial transport.

Ximena G Arceo, Elena F Koslover, Brian M Zid, Aidan I Brown.

For many nuclear-encoded mitochondrial genes, mRNA localizes to the mitochondrial surface co-translationally, aided by the association of a mitochondrial targeting sequence (MTS) on the nascent peptide with the mitochondrial import complex. For a subset of these co-translationally localized mRNAs, their localization is dependent on the metabolic state of the cell, while others are constitutively localized. To explore the differences between these two mRNA types we developed a stochastic, quantitative model for MTS-mediated mRNA localization to mitochondria in yeast cells. This model includes translation, applying gene-specific kinetics derived from experimental data; and diffusion in the cytosol. Even though both mRNA types are co-translationally localized we found that the steady state number, or density, of ribosomes along an mRNA was insufficient to differentiate the two mRNA types. Instead, conditionally-localized mRNAs have faster translation kinetics which modulate localization in combination with changes to diffusive search kinetics across metabolic states. Our model also suggests that the MTS requires a maturation time to become competent to bind mitochondria. Our work indicates that yeast cells can regulate mRNA localization to mitochondria by controlling mitochondrial volume fraction (influencing diffusive search times) and gene translation kinetics (adjusting mRNA binding competence) without the need for mRNA-specific binding proteins. These results shed light on both global and gene-specific mechanisms that enable cells to alter mRNA localization in response to changing metabolic conditions.

DOI: https://doi.org/10.1371/journal.pcbi.1010413
J Biol Chem. 2022 Aug 10. pii: S0021-9258(22)00811-0. [Epub ahead of print] 102368

Human eukaryotic initiation factor 4E (eIF4E) and the nucleotide-bound state of eIF4A regulate eIF4F binding to RNA.

Mario Servulo Izidoro, Masaaki Sokabe, Nancy Villa, William C Merrick, Christopher S Fraser.

  During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate the binding of eIF4E to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. Additionally, we show the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation constant observed in the presence versus absence of ATP. By monitoring uncapped RNA dissociation in real time, we reveal ATP reduces the dissociation rate constant of RNA for eIF4F by ∼4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a highly dynamic state that has very fast association and dissociation rates from RNA. Monitoring the kinetic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.

Keywords:  RNA; cooperativity; eIF4A; eIF4E; eIF4F; eIF4G; translation initiation

DOI:  https://doi.org/10.1016/j.jbc.2022.102368
Mol Biol Cell. 2022 Aug 17. mbcE21090437

Evidence for low-level translation in human erythrocytes.

Sangeetha Devi Kumar, Debaleena Kar, Md Noor Akhtar, Belinda Willard, Debadrita Roy, Tanweer Hussain, Purusharth I Rajyaguru, Sandeep M Eswarappa.

It is generally believed that human mature erythrocytes do not possess functional ribosomes, and therefore cannot synthesize proteins. However, the absence of translation is not consistent with the long lifespan of mature erythrocytes. They stay viable and functional for about 115 days in the circulatory system. Here, using highly pure preparation of human mature erythrocytes, we demonstrate the presence of translation by polysome profiling, [35S]methionine labelling and RiboPuromycylation. [35S]methionine labelling revealed that the translation in mature erythrocytes is about 10% of that observed in reticulocytes. We could observe polysomes by transmission electron microscopy in these cells. RNA-seq and quantitative RT-PCR performed on polysome fraction of these cells revealed that HBA (alpha globin) and HBB (beta globin) transcripts are translated. Using luciferase-based reporter assay and mutational studies, we show that the sequence of the 5' untranslated region is crucial for the translation of these transcripts. Furthermore, mature erythrocytes showed reduced expression of globin proteins (alpha and beta) when treated with translation inhibitors. Overall, we provide multiple lines of evidence for translation of globin mRNAs in human mature erythrocytes.

DOI: https://doi.org/10.1091/mbc.E21-09-0437
Nature. 2022 Aug 17.

How much virus does a person with COVID exhale? New research has answers.

McKenzie Prillaman.



Keywords:  Medical research; SARS-CoV-2; Virology

DOI:  https://doi.org/10.1038/d41586-022-02202-z