DHX36 prevents the accumulation of translationally inactive mRNAs with G4-structures in untranslated regions

Translation efficiency can be affected by mRNA stability and secondary structures, including G-quadruplex structures (G4s). The highly conserved DEAH-box helicase DHX36/RHAU resolves G4s on DNA and RNA in vitro, however a systems-wide analysis of DHX36 targets and function is lacking. We map globally DHX36 binding to RNA in human cell lines and find it preferentially interacting with G-rich and G4-forming sequences on more than 4500 mRNAs. While DHX36 knockout (KO) results in a significant increase in target mRNA abundance, ribosome occupancy and protein output from these targets decrease, suggesting that they were rendered translationally incompetent. Considering that DHX36 targets, harboring G4s, preferentially localize in stress granules, and that DHX36 KO results in increased SG formation and protein kinase R (PKR/EIF2AK2) phosphorylation, we speculate that DHX36 is involved in resolution of rG4 induced cellular stress

Proximity‐CLIP and Expedited Non‐Radioactive Library Preparation of Small RNA Footprints for Next‐Generation Sequencing

Along their life cycle most RNAs move between several cellular environments where they associate with different RNA Binding Proteins (RBPs). Reciprocally, a significant portion of RBPs reside in more than a single cellular compartment, where they can interact with discrete RNAs and even exert distinct biological roles. Proximity-CLIP combines proximity biotinylation of proteins with photoactivatable ribonucleoside-enhanced protein-RNA crosslinking to simultaneously profile the proteome including RBPs and the RBP-bound transcriptome in any given subcellular compartment. Here we provide a detailed experimental protocol for Proximity-CLIP with a simplified non-radioactive, small RNA cDNA library preparation protocol

Epigenetic repression of antiviral genes by SARS-CoV-2 NSP1.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades the innate immune machinery through multiple viral proteins, including nonstructural protein 1 (NSP1). While NSP1 is known to suppress translation of host mRNAs, the mechanisms underlying its immune evasion properties remain elusive. By integrating RNA-seq, ribosome footprinting, and ChIP-seq in A549 cells we found that NSP1 predominantly represses transcription of immune-related genes by favoring Histone 3 Lysine 9 dimethylation (H3K9me2). G9a/GLP H3K9 methyltransferase inhibitor UNC0638 restored expression of antiviral genes and restricted SARS-CoV-2 replication. Our multi-omics study unravels an epigenetic mechanism underlying host immune evasion by SARS-CoV-2 NSP1. Elucidating the factors involved in this phenomenon, may have implications for understanding and treating viral infections and other immunomodulatory diseases

Evidence for a cytoplasmic pool of ribosome-free mRNAs encoding inner membrane proteins in Escherichia coli.

Translation-independent mRNA localization represents an emerging concept in cell biology. In Escherichia coli, mRNAs encoding integral membrane proteins (MPRs) are targeted to the membrane where they are translated by membrane associated ribosomes and the produced proteins are inserted into the membrane co-translationally. In order to better understand aspects of the biogenesis and localization of MPRs, we investigated their subcellular distribution using cell fractionation, RNA-seq and qPCR. The results show that MPRs are overrepresented in the membrane fraction, as expected, and depletion of the signal recognition particle-receptor, FtsY reduced the amounts of all mRNAs on the membrane. Surprisingly, however, MPRs were also found relatively abundant in the soluble ribosome-free fraction and their amount in this fraction is increased upon overexpression of CspE, which was recently shown to interact with MPRs. CspE also conferred a positive effect on the membrane-expression of integral membrane proteins. We discuss the possibility that the effects of CspE overexpression may link the intriguing subcellular localization of MPRs to the cytosolic ribosome-free fraction with their translation into membrane proteins and that the ribosome-free pool of MPRs may represent a stage during their targeting to the membrane, which precedes translation

High throughput sequencing of endogenous RNAs that co-purify with 6His-CspE.

<p><b>(A)</b> Wild type <i>E</i>. <i>coli</i> expressing CspE-6His were disrupted in the presence of either 2 mM or 15 mM [Mg²⁺] (top and bottom panels, respectively) and the total cell extracts were subjected to metal affinity chromatography using Talon resin. RNA was prepared from the total cell extracts and the imidazole-eluted material (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134413#pone.0134413.g003" target="_blank">Fig 3</a>) and subjected to high throughput sequencing. The amount of CspE-6His bound MPRs (left panels) and CPRs (right panels) is plotted as a function of the amount of the same mRNAs in the total extract. <b>(B)</b> CspE-binding of all detected mRNAs was calculated as [RPKM_CspE-bound / RPKM_extract]. The quota of MPRs and CPRs in each 10^th percentile along the CspE-association landscape is presented as a moving average plot. <b>(C)</b> This panel shows the CspE-binding values in the presence of 2 mM [Mg²⁺] for selected MPRs and CPRs that were similarly analyzed by qPCR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134413#pone.0134413.g003" target="_blank">Fig 3D</a> for comparison). <b>(D)</b> An independent experiment that shows the CspE-binding values for selected MPRs and CPRs in the presence of 15 mM [Mg²⁺].</p

DHX36 binding at G-rich sites in mRNA untranslated regions promotes translation

ABSTRACTTranslation efficiency can be affected by mRNA stability and secondary structures, including so-called G-quadruplex (G4) structures. The highly conserved and essential DEAH-box helicase DHX36/RHAU is able to resolve G4 structures on DNA and RNA in vitro, however a system-wide analysis of DHX36 targets and function is lacking. We globally mapped DHX36 occupancy in human cell lines and found that it preferentially binds to G-rich sequences in the coding sequences (CDS) and 5' and 3' untranslated regions (UTR) of more than 4,500 mRNAs. Functional analyses, including RNA sequencing, ribosome footprinting, and quantitative mass spectrometry revealed that DHX36 decreased target mRNA stability. However, target mRNA accumulation in DHX36 KO cells did not lead to a significant increase in ribosome footprints or protein output indicating that they were translationally incompetent. We hypothesize that DHX36 resolves G4 and other structures that interfere with efficient translation initiation.</jats:p

Characterization of 4 model untranslatable RNAs.

<p><b>(A)</b> Schematic representation of the Ra-Rd encoding genes [see text, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134413#pone.0134413.s001" target="_blank">S1 Fig</a>]. <b>(B)</b> Uracil content of the model transcripts, utilizing a sliding window of 55 nucleotides as calculated by the software DNA Strider. <b>(C)</b> Wild type <i>E</i>. <i>coli</i> expressing Ra or Rb were disrupted by sonication and cell extracts were fractionated by sucrose density gradient (a representative gradient is shown). The gradient fractions were analyzed for RNA content (A₂₆₀). The indicated free and ribosomal fractions were pooled. <b>(D)</b> The contents of the indicated R transcripts and endogenous, translatable transcripts encoding PrfA and RpoD as controls, were measured by qPCR in the pooled free and ribosomal fractions. Error bars indicate SEM (n = 3).</p

Characterization of 4 model untranslatable RNAs.

<p><b>(A)</b> Schematic representation of the Ra-Rd encoding genes [see text, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134413#pone.0134413.s001" target="_blank">S1 Fig</a>]. <b>(B)</b> Uracil content of the model transcripts, utilizing a sliding window of 55 nucleotides as calculated by the software DNA Strider. <b>(C)</b> Wild type <i>E</i>. <i>coli</i> expressing Ra or Rb were disrupted by sonication and cell extracts were fractionated by sucrose density gradient (a representative gradient is shown). The gradient fractions were analyzed for RNA content (A₂₆₀). The indicated free and ribosomal fractions were pooled. <b>(D)</b> The contents of the indicated R transcripts and endogenous, translatable transcripts encoding PrfA and RpoD as controls, were measured by qPCR in the pooled free and ribosomal fractions. Error bars indicate SEM (n = 3).</p

Model Uracil-Rich RNAs and Membrane Protein mRNAs Interact Specifically with Cold Shock Proteins in <i>Escherichia coli</i>

<div><p>Are integral membrane protein-encoding mRNAs (MPRs) different from other mRNAs such as those encoding cytosolic mRNAs (CPRs)? This is implied from the emerging concept that MPRs are specifically recognized and delivered to membrane-bound ribosomes in a translation-independent manner. MPRs might be recognized through uracil-rich segments that encode hydrophobic transmembrane helices. To investigate this hypothesis, we designed DNA sequences encoding model untranslatable transcripts that mimic MPRs or CPRs. By utilizing <i>in vitro</i>-synthesized biotinylated RNAs mixed with <i>Escherichia coli</i> extracts, we identified a highly specific interaction that takes place between transcripts that mimic MPRs and the cold shock proteins CspE and CspC, which are normally expressed under physiological conditions. Co-purification studies with <i>E</i>. <i>coli</i> expressing 6His-tagged CspE or CspC confirmed that the specific interaction occurs <i>in vivo</i> not only with the model uracil-rich untranslatable transcripts but also with endogenous MPRs. Our results suggest that the evolutionarily conserved cold shock proteins may have a role, possibly as promiscuous chaperons, in the biogenesis of MPRs.</p></div