RACK1 Is a Ribosome Scaffold Protein for β-actin mRNA/ZBP1 Complex

In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including β-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the β-actin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs

A Comparison of the Crystal Structures of Eukaryotic and Bacterial SSU Ribosomal RNAs Reveals Common Structural Features in the Hypervariable Regions

While the majority of the ribosomal RNA structure is conserved in the three major domains of life – archaea, bacteria, and eukaryotes, specific regions of the rRNA structure are unique to at least one of these three primary forms of life. In particular, the comparative secondary structure for the eukaryotic SSU rRNA contains several regions that are different from the analogous regions in the bacteria. Our detailed analysis of two recently determined eukaryotic 40S ribosomal crystal structures, Tetrahymena thermophila and Saccharomyces cerevisiae, and the comparison of these results with the bacterial Thermus thermophilus 30S ribosomal crystal structure: (1) revealed that the vast majority of the comparative structure model for the eukaryotic SSU rRNA is substantiated, including the secondary structure that is similar to both bacteria and archaea as well as specific for the eukaryotes, (2) resolved the secondary structure for regions of the eukaryotic SSU rRNA that were not determined with comparative methods, (3) identified eukaryotic helices that are equivalent to the bacterial helices in several of the hypervariable regions, (4) revealed that, while the coaxially stacked compound helix in the 540 region in the central domain maintains the constant length of 10 base pairs, its two constituent helices contain 5+5 bp rather than the 6+4 bp predicted with comparative analysis of archaeal and eukaryotic SSU rRNAs.This work was supported by grants from the National Institutes of Health (R01GM067317) and the Welch Foundation (F-1427). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Center for Computational Biology and Bioinformatic

Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM

In recent work with large high symmetry viruses, single particle electron cryomicroscopy (cryoEM) has reached the milestone of determining near atomic resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains extraordinarily challenging. Using a newly developed single electron counting detector, we confirm that electron beam induced motion significantly degrades resolution and, importantly, show how the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy. Thus, intrinsic image information can be restored to high resolution (Thon rings visible to ~3 Å). Using this approach we determined a 3.3 Å resolution structure of a ~700 kDa protein with D7 symmetry showing clear side chain density. Our method greatly enhances image quality and data acquisition efficiency - key bottlenecks in applying near atomic resolution cryoEM to a broad range of protein samples