A cap 0-dependent mRNA capture method to analyze the yeast transcriptome

Analysis of the protein coding transcriptome by the RNA sequencing requires either enrichment of the desired fraction of coding transcripts or depletion of the abundant non-coding fraction consisting mainly of rRNA. We propose an alternative mRNA enrichment strategy based on the RNA-binding properties of the human IFIT1, an antiviral protein recognizing cap 0 RNA. Here, we compare for Saccharomyces cerevisiae an IFIT1-based mRNA pull-down with yeast targeted rRNA depletion by the RiboMinus method. IFIT1-based RNA capture depletes rRNA more effectively, producing high quality RNA-seq data with an excellent coverage of the protein coding transcriptome, while depleting cap-less transcripts such as mitochondrial or some non-coding RNAs. We propose IFIT1 as a cost effective and versatile tool to prepare mRNA libraries for a variety of organisms with cap 0 mRNA ends, including diverse plants, fungi and eukaryotic microbes

IFITs: Emerging Roles As Key Anti-Viral Proteins

Interferon-induced protein with tetratricopeptide repeats (IFITs) are a family of proteins which are strongly induced downstream of type I interferon (IFN) signaling. The molecular mechanism of IFIT anti-viral activity has been studied in some detail, including the recently discovered direct binding of viral nucleic acid, the binding to viral and host proteins, and the possible involvement in anti-viral immune signal propagation. The unique structures of some members of the IFIT family have been solved to reveal an internal pocket for non-sequence-specific, but conformation- and modification-specific, nucleic acid binding. This review will focus on recent discoveries which link IFITs to the anti-viral response, intrinsic to the innate immune system

The Repeating, Modular Architecture of the HtrA Proteases

A conserved, 26-residue sequence [AA(X2)[A/G][G/L](X2)GDV[I/L](X2)[V/L]NGE(X1)V(X6)] and corresponding structure repeating module were identified within the HtrA protease family using a non-redundant set (N = 20) of publicly available structures. While the repeats themselves were far from sequence perfect, they had notable conservation to a statistically significant level. Three or more repetitions were identified within each protein despite being statistically expected to randomly occur only once per 1031 residues. This sequence repeat was associated with a six stranded antiparallel β-barrel module, two of which are present in the core of the structures of the PA clan of serine proteases, while a modified version of this module could be identified in the PDZ-like domains. Automated structural alignment methods had difficulties in superimposing these β-barrels, but the use of a target human HtrA2 structure showed that these modules had an average RMSD across the set of structures of less than 2 Å (mean and median). Our findings support Dayhoff’s hypothesis that complex proteins arose through duplication of simpler peptide motifs and domains

The Seed Region of a Small RNA Drives the Controlled Destruction of the Target mRNA by the Endoribonuclease RNase E

Numerous small non-coding RNAs (sRNAs) in bacteria modulate rates of translation initiation and degradation of target mRNAs, which they recognize through base-pairing facilitated by the RNA chaperone Hfq. Recent evidence indicates that the ternary complex of Hfq, sRNA and mRNA guides endoribonuclease RNase E to initiate turnover of both the RNAs. We show that a sRNA not only guides RNase E to a defined site in a target RNA, but also allosterically activates the enzyme by presenting a monophosphate group at the 5′-end of the cognate-pairing “seed.” Moreover, in the absence of the target the 5′-monophosphate makes the sRNA seed region vulnerable to an attack by RNase E against which Hfq confers no protection. These results suggest that the chemical signature and pairing status of the sRNA seed region may help to both ‘proofread’ recognition and activate mRNA cleavage, as part of a dynamic process involving cooperation of RNA, Hfq and RNase E

The regulatory protein RraA modulates RNA-binding and helicase activities of the E. coli RNA degradosome

The Escherichia coli endoribonuclease RNase E is an essential enzyme having key roles in mRNA turnover and the processing of several structured RNA precursors, and it provides the scaffold to assemble the multienzyme RNA degradosome. The activity of RNase E is inhibited by the protein RraA, which can interact with the ribonuclease's degradosome-scaffolding domain. Here, we report that RraA can bind to the RNA helicase component of the degradosome (RhlB) and the two RNA-binding sites in the degradosome-scaffolding domain of RNase E. In the presence of ATP, the helicase can facilitate the exchange of RraA for RNA stably bound to the degradosome. Our data suggest that RraA can affect multiple components of the RNA degradosome in a dynamic, energy-dependent equilibrium. The multidentate interactions of RraA impede the RNA-binding and ribonuclease activities of the degradosome and may result in complex modulation and rerouting of degradosome activity

Multi-OMICs landscape of SARS-CoV-2-induced host responses in human lung epithelial cells

Summary: COVID-19 pandemic continues to remain a global health concern owing to the emergence of newer variants. Several multi-Omics studies have produced extensive evidence on host-pathogen interactions and potential therapeutic targets. Nonetheless, an increased understanding of host signaling networks regulated by post-translational modifications and their ensuing effect on the cellular dynamics is critical to expanding the current knowledge on SARS-CoV-2 infections. Through an unbiased transcriptomics, proteomics, acetylomics, phosphoproteomics, and exometabolome analysis of a lung-derived human cell line, we show that SARS-CoV-2 Norway/Trondheim-S15 strain induces time-dependent alterations in the induction of type I IFN response, activation of DNA damage response, dysregulated Hippo signaling, among others. We identified interplay of phosphorylation and acetylation dynamics on host proteins and its effect on the altered release of metabolites, especially organic acids and ketone bodies. Together, our findings serve as a resource of potential targets that can aid in designing novel host-directed therapeutic strategies

The free energy of conformational changes of the A-loop.

<p>The free energy surfaces of the A-loop transition from open (active-like) to closed (inactive-like) conformation in case of the ABL catalytic domain alone (left) and in presence of the SH2 regulatory domain in the “top-hat” conformation as a function of the contact map distances to the respective reference structures. For the deepest minima a representative structure is also shown below with the CD colored in blue, the SH2 in green, the A-loop in yellow and the aC-helix in red.</p

List of c-Abl point mutants investigated in this study with summary of the effect of mutations.

<p>Mutants were tested for c-Abl activity via immunoblotting of HEK293 lysates or immunoprecipitates (IP) and via kinase activity assay. The numbering of residues is in agreement with the sequence of the isoform Ib. Activity scoring (effect of the mutation):, nt, not tested, − inactive (mutation disruptive), + weakly active (mutation mildly disruptive), ++ activity similar to wild-type (mutation neutral), +++ hyperactive (mutation activating) (See also Supplemental Table S1).</p><p>List of c-Abl point mutants investigated in this study with summary of the effect of mutations.</p

Domain organization and crystal structures of Abl kinase.

<p><b>A</b> The c-Abl isoform Ib is characterized by myristoylation (Myr) on Gly-2 of the N-terminal capping region (cap). The tyrosine kinase domain is preceded by the SH3 and SH2 domains and a connecting linker. The last exon region contains nuclear localization signals and a C-terminal actin binding domain (ABD). <b>B</b> In the down-regulated state (PDB entry 2FO0), the SH2 domain binds the C-lobe of the kinase domain, the myristate is bound in its cognate pocket and the SH3 domain binds the SH2-CD linker. <b>C</b> In the active “top-hat” conformation (PDB entry 1OPL), the SH2 domain moves to interact with the N-lobe of the kinase domain. The αC helix and the activation loop are highlighted in red and pink, respectively. <b>D</b> Positions of the most important point mutations at the SH2-CD interface and in the β3-αC loop.</p

Effect of point mutations on the kinase activity of c-Abl.

<p>Abl proteins were immunoprecipitated and assayed for phosphorylation of an optimal Abl substrate peptide. The kinase activity was normalized to the amount of protein and to the activity of the wild-type CD or SH2-CD construct. <b>A</b> M297L was found to decrease kinase activity, whereas M297G had an activating effect in the absence of the SH2 domain. The kinase-dead mutant D382N presented no activity, and the known inactivating (I164E) and activating (T231R) mutations also show, accordingly, a decreased and increased activity. <b>B</b> The M297G mutant is significantly more active in the context of the isolated kinase domain, but not in the presence of the SH2 domain. The error bars are standard deviations from biological quadruplicates (n = 4, **P<0.01, Student <i>t</i> test). <b>C</b> Mutations E294P and E294P V299P have an activating effect. The reactions were performed at 37°C. <b>D</b> The activity of wild-type and E294P V299P Abl proteins was measured at increasing substrate concentrations and 25 µM ATP. <b>E</b> Y339G substitution is neutral, whereas Y339P diminishes Abl activity. The effect is seen both at 24°C and at an elevated temperature. <b>F, G</b> The ratio of the kinase activity at the elevated and room temperatures. Except for Y339P, the SH2-CD proteins retain their activity at the elevated temperature, whereas the activity of the CD constructs is reduced at least 2-fold. The error bars are standard deviations from technical triplicates except for (B). (See also Figures S4 and S5).</p