mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding [preprint]

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Forster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation

Recent advances and current trends in cryo-electron microscopy

All steps of cryogenic electron-microscopy (cryo-EM) workflows have rapidly evolved over the last decade. Advances in both single-particle analysis (SPA) cryo-EM and cryo-electron tomography (cryo-ET) have facilitated the determination of high-resolution biomolecular structures that are not tractable with other methods. However, challenges remain. For SPA, these include improved resolution in an additional dimension: time. For cryo-ET, these include accessing difficult-to-image areas of a cell and finding rare molecules. Finally, there is a need for automated and faster workflows, as many projects are limited by throughput. Here, we review current developments in SPA cryo-EM and cryo-ET that push these boundaries. Collectively, these advances are poised to propel our spatial and temporal understanding of macromolecular processes

The space between notes: emerging roles for translationally silent ribosomes

In addition to their central functions in translation, ribosomes can adopt inactive structures that are fully assembled yet devoid of mRNA. We describe how the abundance of idle eukaryotic ribosomes is influenced by a broad range of biological conditions spanning viral infection, nutrient deprivation, and developmental cues. Vacant ribosomes may provide a means to exclude ribosomes from translation while also shielding them from degradation, and the variable identity of factors that occlude ribosomes may impart distinct functionality. We propose that regulated changes in the balance of idle and active ribosomes provides a means to fine-tune translation. We provide an overview of idle ribosomes, describe what is known regarding their function, and highlight questions that may clarify their biological roles

Cancer-Relevant Splicing Factor CAPERalpha Engages the Essential Splicing Factor SF3b155 in a Specific Ternary Complex

U2AF Homology Motifs (UHMs) mediate protein-protein interactions with U2AF Ligand Motifs (ULMs) of pre-mRNA splicing factors. The UHM-containing alternative splicing factor CAPERalpha regulates splicing of tumor-promoting VEGF isoforms, yet the molecular target of the CAPERalpha UHM is unknown. Here, we present structures of the CAPERalpha UHM bound to a representative SF3b155 ULM at 1.7 A resolution, and for comparison, in the absence of ligand at 2.2 A resolution. The prototypical UHM/ULM interactions authenticate CAPERalpha as a bona fide member of the UHM-family of proteins. We identify SF3b155 as the relevant ULM-containing partner of full-length CAPERalpha in human cell extracts. Isothermal titration calorimetry comparisons of the purified CAPERalpha UHM binding known ULM-containing proteins demonstrate that high affinity interactions depend on the presence of an intact, intrinsically-unstructured SF3b155 domain containing seven ULM-like motifs. The interplay among bound CAPERalpha molecules gives rise to the appearance of two high affinity sites in the SF3b155 ULM-containing domain. In conjunction with the previously-identified, UHM/ULM-mediated complexes of U2AF65 and SPF45 with SF3b155, this work demonstrates the capacity of SF3b155 to offer a platform for coordinated recruitment of UHM-containing splicing factors

SF1 Phosphorylation Enhances Specific Binding to U2AF 65 and Reduces Binding to 3′-Splice-Site RNA

International audienceSplicing factor 1 (SF1) recognizes 3' splice sites of the major class of introns as a ternary complex with U2AF65 and U2AF35 splicing factors. A conserved SPSP motif in a coiled-coil domain of SF1 is highly phosphorylated in proliferating human cells and is required for cell proliferation. The UHM kinase 1 (UHMK1), also called KIS, double-phosphorylates both serines of this SF1 motif. Here, we use isothermal titration calorimetry to demonstrate that UHMK1 phosphorylation of the SF1 SPSP motif slightly enhances specific binding of phospho-SF1 to its cognate U2AF65 protein partner. Conversely, quantitative fluorescence anisotropy RNA binding assays and isothermal titration calorimetry experiments establish that double-SPSP phosphorylation reduces phospho-SF1 and phospho-SF1-U2AF65 binding affinities for either optimal or suboptimal splice-site RNAs. Domain-substitution and mutagenesis experiments further demonstrate that arginines surrounding the phosphorylated SF1 loop are required for cooperative 3' splice site recognition by the SF1-U2AF65 complex (where cooperativity is defined as a nonadditive increase in RNA binding by the protein complex relative to the individual proteins). In the context of local, intracellular concentrations, the subtle effects of SF1 phosphorylation on its associations with U2AF65 and splice-site RNAs are likely to influence pre-mRNA splicing. However, considering roles for SF1 in pre-mRNA retention and transcriptional repression, as well as in splicing, future comprehensive investigations are needed to fully explain the requirement for SF1 SPSP phosphorylation in proliferating human cells

Functionally distinct roles for eEF2K in the control of ribosome availability and p-body abundance

Processing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splitting in vitro. Collectively, the data suggest that eEF2K defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons

Cryo-EM fibril structures from systemic AA amyloidosis reveal the species complementarity of pathological amyloids

Systemic AA amyloidosis is a worldwide occurring protein misfolding disease of humans and animals. It arises from the formation of amyloid fibrils from the acute phase protein serum amyloid A. Here, we report the purification and electron cryo-microscopy analysis of amyloid fibrils from a mouse and a human patient with systemic AA amyloidosis. The obtained resolutions are 3.0 angstrom and 2.7 angstrom for the murine and human fibril, respectively. The two fibrils differ in fundamental properties, such as presence of right-hand or left-hand twisted cross-beta sheets and overall fold of the fibril proteins. Yet, both proteins adopt highly similar beta-arch conformations within the N-terminal similar to 21 residues. Our data demonstrate the importance of the fibril protein N-terminus for the stability of the analyzed amyloid fibril morphologies and suggest strategies of combating this disease by interfering with specific fibril polymorphs