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RNA footprinting by hydroxyl radical cleavage provides 'snapshots' of RNA tertiary structure or protein interactions that bury the RNA backbone. Generation of hydroxyl radicals with a high-flux synchrotron X-ray beam provides analysis on a short timescale (5-100 msec), which enables the structures of folding intermediates or other transient conformational states to be determined in biochemical solutions or cells. This article provides protocols for using synchrotron beamlines for hydroxyl radical footprinting. © 2018 by John Wiley & Sons, Inc.

Polyadenylylation of mRNA appears to be common in the mycobacteria. RNA polyadenylylation controls plasmid replication, modulates RNA stability and may participate in mRNA translation.

Research has been conducted on the salicylic acid. The role of this acid in iron metabolism has been investigated.

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. [ABSTRACT FROM AUTHOR]

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 chromatin immunoprecipitation (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, whereas 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. [ABSTRACT FROM AUTHOR]

Abstract: To study the role of sequence and topology in RNA folding, we determined the kinetic folding pathways of two circularly permuted variants of the Tetrahymena group I ribozyme, using time-resolved hydroxyl radical footprinting. Circular permutation changes the distance between interacting residues in the primary sequence, without changing the native structure of the RNA. In the natural ribozyme, tertiary interactions in the P4-P6 domain form in 1 s, while interactions in the P3-P9 form in 1–3 min at 42 °C. Permutation of the 5′ end to G111 in the P4 helix allowed the stable P4-P6 domain to fold in 200 ms at 30 °C, five times faster than in the wild-type RNA, while the other domains folded five times more slowly (5–8 min). By contrast, circular permutation of the 5′ end to G303 in J8/7 decreased the folding rate of the P4-P6 domain. In this permuted RNA, regions joining P2, P3 and P4 were protected in 500 ms, while the P3-P9 domain was 60–80% folded within 30 s. RNase T1 digestion and FMN photocleavage showed that circular permutation of the RNA sequence alters the initial ensemble of secondary structures, thereby changing the tertiary folding pathways. Our results show that the natural 5′-to-3′ order of the structural domains in group I ribozymes optimizes structural communication between tertiary domains and promotes self-assembly of the catalytic center. [Copyright &y& Elsevier]

Evolution of the ribosome from an RNA catalyst suggests that the intrinsic folding pathway of the rRNA dictates the hierarchy of ribosome assembly. To address this possibility, we probed the tertiary folding pathway of the 5′ domain of the Escherichia coli 16S rRNA at 20ms intervals using X-ray-dependent hydroxyl radical footprinting. Comparison with crystallographic structures and footprinting reactions on native 30S ribosomes showed that the RNA formed all of the predicted tertiary interactions in the absence of proteins. In 20mM MgCl2, many tertiary interactions appeared within 20ms. By contrast, interactions between H6, H15 and H17 near the spur of the 30S ribosome evolved over several minutes, likely due to mispairing of a central helix junction. The kinetic folding pathway of the RNA corresponded to the expected order of protein binding, suggesting that the RNA folding pathway forms the basis for early steps of ribosome assembly. [Copyright &y& Elsevier]

Analyzes the coding sequence and 3' untranslated region (UTR) of the 14-3-3 gene of the euryhaline teleost Fundulus heteroclitus. Primary structure of the 14-3-3 protein; Osmotic regulation and tissue specificity expression; Three-dimensional structure; Amino acid sequence; Role of the 14-3-3 in the regulation of ion transport.

Chemical footprinting methods are widely used to probe the solution structures of nucleic acids and their complexes. Among the many available modifying reagents, hydroxyl radical is exceptional in its ability to provide nucleotide-level information on the solvent accessibility of the nucleic acid backbone. Until recently, hydroxyl radical footprinting has been limited to in vitro experiments. We describe the use of synchrotron X-radiation to generate hydroxyl radicals within cells for effective footprinting of RNA-protein complexes in vivo. This technique gives results that are consistent with in vitro footprinting experiments, with differences reflecting apparent structural changes to the RNA in vivo., (Copyright © 2009 Elsevier Inc. All rights reserved.)

Coordinate regulation of the ribosomal protein genes is entrusted to a number of signal transduction pathways that can abruptly induce or silence the ribosomal genes. We have uncovered a cellular model system, which selectively induces the ribosomal protein S25 gene in hepatoma cells that are stressed by nutrient deprivation. Our results indicate that p53 along with two other identified proteins, MTF-1 and La, post-transcriptionally regulate the synthesis of the S25 protein by controlling the nuclear export of the stress-induced S25 mRNA. This system is unique in that the nuclear-retained S25 mRNA is exported to the cytosol only upon replenishment or alternatively after prolonged starvation to participate in a p53-mediated apoptotic sequence of events. This p53-dependent survival or death pathway involves a previously unreported protein relationship among these three actors, one of which, MTF-1, has not yet been shown to have RNA-binding characteristics.