SummaryRapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. 1,2 How the 16S rRNA and 20 proteins that make up the 30S ribosomal subunit assemble faithfully in a few minutes remains a challenging problem, in part because real-time data on the earliest stages of assembly are lacking. Here, we show that 30S assembly nucleates concurrently from different points along the rRNA, by providing snapshots of individual RNA and protein interactions as they emerge in real time. Time-resolved hydroxyl radical footprinting 3 was used to map changes in the structure of the rRNA within 20 ms after addition of total 30S proteins. Helix junctions in each domain fold within 100 ms. By contrast, interactions surrounding the decoding site and between the 5′, central and 3′ domains require 2-200 seconds to form. Surprisingly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA-protein encounter complexes refold during assembly. While early steps in assembly are linked to intrinsically stable rRNA structure, later steps correspond to regions of induced fit between the proteins and the rRNA.Nomura and colleagues demonstrated that hierarchical addition of ribosomal proteins to the 16S rRNA produces cooperativity, 4 that is due to protein-induced structural changes in the 16S rRNA rather than direct contacts between proteins. 5 As the rRNA becomes more structured as proteins join the complex, assembly is coupled to the folding pathway of the rRNA. 6,7 The simplest kinetic model for 30S assembly is that regions of the 16S rRNA contacted by primary assembly proteins fold earliest, while helices stabilized by tertiary assembly proteins fold last. If assembly is strictly sequential, each subdomain of the rRNA will fold within a distinct time, producing a limited set of intermediate complexes.Alternatively, the rate of protein binding may initially depend on the stochastic probability of forming locally stable rRNA and protein interactions, with progression to later intermediates depending on propagation of this conformational order to neighboring regions in the rRNA. If more than one region of the naked rRNA can fold, assembly is expected to nucleate from many places at once, producing an ensemble of reconstitution intermediates and multi-stage assembly kinetics. 8 *Corresponding author: E-mail: swoodson@jhu.edu, tel. +001-410-516-2015, FAX +001-410-516-4118 To visualize the intermediates of 30S ribosome assembly, the structure of the 16S rRNA was probed by time-resolved X-ray hydroxyl radical footprinting (Figure 1). The extent of RNA cleavage correlates with backbone exposure. 9 Thus, this method probes individual tertiary contacts in the rRNA as well as protein interactions that bury the rRNA backbone. Previous efforts to map the conformational changes in the 16S rRNA during 30S ribosome assembly used low temperature or subsets of proteins to stall assembly at specific stages. 10,11 We took advantage of the...

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We used a high flux synchrotron X-ray beam to map the structure of 16S rRNA and RNase P in viable bacteria in situ. A 300 ms exposure to the X-ray beam was sufficient for optimal cleavage of the phosphodiester backbone. The in vivo footprints of the 16S rRNA in frozen cells were similar to those obtained in vitro and were consistent with the predicted accessibility of the RNA backbone to hydroxyl radical. Protection or enhanced cleavage of certain nucleotides in vivo can be explained by interactions with tRNA and perturbation of the subunit interface. Thus, short exposures to a synchrotron X-ray beam can footprint the tertiary structure and protein contacts of RNA–protein complexes with nucleotide resolution in living cells.

Injury response in the peripheral nervous system (PNS) is characterized by rapid alterations in the genetic program of Schwann cells. However, the epigenetic mechanisms modulating these changes remain elusive. Here we show that sciatic nerve injury in mice induces a cohort of 22 miRNAs, which coordinate Schwann cell differentiation and dedifferentiation through a combinatorial modulation of their positive and negative gene regulators. These miRNAs and their targeted mRNAs form functional complexes with the Argonaute-2 protein to mediate post-transcriptional gene silencing. MiR-138 and miR-709 show the highest affinity amongst the cohort, for binding and regulation of Egr2, Sox-2 and c-Jun expression following injury. Moreover, miR-709 participates in the formation of epigenetic silencing complexes with H3K27me3 and Argonaute-1 to induce transcriptional gene silencing of the Egr2 promoter. Collectively, we identified a discrete cohort of miRNAs as the central epigenetic regulators of the transition between differentiation and dedifferentiation during the acute phase of PNS injury.

Reciprocal interactions between glia and neurons are essential for the proper organization and function of the nervous system. Recently, the interaction between ErbB receptors (ErbB2 and ErbB3) on the surface of Schwann cells and neuronal Neuregulin-1 (NRG1) has emerged as the pivotal signal that controls Schwann cell development, association with axons, and myelination. To understand the function of NRG1-ErbB2/3 signaling axis in adult Schwann cell biology we are studying the specific role of ErbB3 receptor tyrosine kinase (RTK) since it is the receptor for NRG1 on the surface of Schwann cells. Here we show that alternative transcription initiation results in the formation of a nuclear variant of ErbB3 (nuc-ErbB3) in rat primary Schwann cells. Nuc-ErbB3 possesses a functional nuclear localization signal sequence and binds to chromatin. Using ChIP-ChIP arrays we identified the promoters that associate with nuc-ErbB3 and clustered the active promoters in Schwann cell gene expression. Nuc-ErbB3 regulates the transcriptional activity of ezrin and HMGB1 promoters while inhibition of nuc-ErbB3 expression results in reduced myelination and altered distribution of ezrin in the nodes of Ranvier. Finally, we reveal that NRG1 regulates the translation of nuc-ErbB3 in rat Schwann cells. For the first time, to our knowledge, we show that alternative transcription initiation from a gene that encodes a RTK is capable to generate a protein variant of the receptor with a distinct role in molecular and cellular regulation. We propose a new concept for the molecular regulation of myelination through the expression and distinct role of nuc-ErbB3.