The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA

Small noncoding RNAs (sRNAs) regulate the response of bacteria to environmental stress in conjunction with the Sm-like RNA binding protein Hfq. DsrA sRNA stimulates translation of the RpoS stress response factor in Escherichia coli by base-pairing with the 5′ leader of the rpoS mRNA and opening a stem–loop that represses translation initiation. We report that rpoS leader sequences upstream of this stem–loop greatly increase the sensitivity of rpoS mRNA to Hfq and DsrA. Native gel mobility shift assays show that Hfq increases the rate of DsrA binding to the full 576 nt rpoS leader as much as 50-fold. By contrast, base-pairing with a 138-nt RNA containing just the repressor stem–loop is accelerated only twofold. Deletion and mutagenesis experiments showed that sensitivity to Hfq requires an upstream AAYAA sequence. Leaders long enough to contain this sequence bind Hfq tightly and form stable ternary complexes with Hfq and DsrA. A model is proposed in which Hfq recruits DsrA to the rpoS mRNA by binding both RNAs, releasing the self-repressing structure in the mRNA. Once base-pairing between DsrA and rpoS mRNA is established, interactions between Hfq and the mRNA may stabilize the RNA complex by removing Hfq from the sRNA.     

A small RNA activates CFA synthase by isoform‐specific mRNA stabilization

Small RNAs use a diversity of well‐characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq‐associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation‐independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot‐exposed, conserved 5′ end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E‐mediated decay in the 5′ untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA‐controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.     

Structures and recognition modes of toll‐like receptors

Toll‐like receptors (TLRs) recognize common structural patterns in diverse microbial molecules and play central roles in the innate immune response. The structures of extracellular domains and their ligand complexes of several TLRs have been determined by X‐ray crystallography. Here, we discuss recent advances on structures and activation mechanisms of TLRs. Despite the differences in interaction areas of ligand with TLRs, the extracellular domains of TLRs all adopt horseshoe‐shaped structures and the overall M‐shape of the TLR–ligand complexes is strikingly similar. The structural rearrangement information of TLRs sheds new light on their ligand‐recognition and ‐activation mechanisms. Proteins 2016; 85:3–9. © 2016 Wiley Periodicals, Inc.     

Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor‐binding specificities in different bacterial species

ClpXP, an AAA+ protease, plays key roles in protein‐quality control and many regulatory processes in bacteria. The N‐terminal domain of the ClpX component of ClpXP is involved in recognition of many protein substrates, either directly or by binding the SspB adaptor protein, which delivers specific classes of substrates for degradation. Despite very limited sequence homology between the E. coli and C. crescentus SspB orthologs, each of these adaptors can deliver substrates to the ClpXP enzyme from the other bacterial species. We show that the ClpX N domain recognizes different sequence determinants in the ClpX‐binding (XB) peptides of C. crescentus SspBα and E. coli SspB. The C. crescentus XB determinants span 10 residues and involve interactions with multiple side chains, whereas the E. coli XB determinants span half as many residues with only a few important side chain contacts. These results demonstrate that the N domain of ClpX functions as a highly versatile platform for peptide recognition, allowing the emergence during evolution of alternative adaptor‐binding specificities. Our results also reveal highly conserved residues in the XB peptides of both E. coli SspB and C. crescentus SspBα that play no detectable role in ClpX‐binding or substrate delivery.     

Low‐density lipoprotein receptor‐related protein 6 regulates alternative pre‐mRNA splicing

Low‐density lipoprotein receptor‐related protein 6 (LRP6) serves as a Wnt coreceptor. Although Wnt/LRP6 signalling is best known for the β‐catenin‐dependent regulation of target genes in tissue development and homeostasis, emerging evidence demonstrates the biological aspects of LRP6 beyond a Wnt coreceptor. Whether LRP6 modulates tissue development in a Wnt/β‐catenin signalling‐independent manner remains unknown. Using a model of striated muscle development, we observed that LRP6 was almost undetectable in proliferating myoblasts, whereas its expression gradually increased in the nucleus of myodifferentiating cells. During myodifferentiation, LRP6 modulated the muscle‐specific splicing of integrin‐β1D and consequent myotube maturation independently of the β‐catenin‐dependent Wnt signalling. Furthermore, we identified that the carboxy‐terminal serine‐rich region in LRP6 bond to the adenine‐rich sequence within alternative exon D (AED) of integrin‐β1 pre‐mRNA, and therefore, elicited AED inclusion when the spliceosome was recruited to the splice site. The interaction of LRP6 with the adenine‐rich sequence was sufficient to overcome AED exclusion by a splicing repressor, polypyrimidine tract binding protein‐1. Besides the integrin‐β1, deep RNA sequencing in different types of cells revealed that the LRP6‐mediated splicing regulation was widespread. Thus, our findings implicate LRP6 as a potential regulator for alternative pre‐mRNA splicing.     

Brucella melitensis 16MΔhfq attenuation confers protection against wild‐type challenge in BALB/c mice

Brucellosis is a globally distributed zoonotic disease that causes animal and human diseases. Although effective, the current Brucella vaccines (Rev.1 and M5‐90) have several drawbacks. The first involves residual virulence for animals and humans and the second is the inability to differentiate natural infection from that caused by vaccination. Therefore, Brucella melitensis 16M hfq mutant (16MΔhfq) was constructed to overcome these drawbacks. Similarly to Rev.1 and M5‐90, 16MΔhfq reduces survival in macrophages and mice and induces strong protective immunity in BALB/c mice. Moreover, these vaccines elicit anti‐Brucella‐specific IgG1 and IgG2a subtype responses and induce secretion of gamma interferon and interleukin‐4. The Hfq antigen also allows serological differentiation between infected and vaccinated animals. These results show that 16MΔhfq is an ideal live attenuated vaccine candidate against virulent Brucella melitensis 16M infection. It will be further evaluated in sheep.     

Analysis of the interaction with the hepatitis C virus mRNA reveals an alternative mode of RNA recognition by the human La protein

Human La protein is an essential factor in the biology of both coding and non-coding RNAs. In the nucleus, La binds primarily to 3' oligoU containing RNAs, while in the cytoplasm La interacts with an array of different mRNAs lacking a 3' UUU(OH) trailer. An example of the latter is the binding of La to the IRES domain IV of the hepatitis C virus (HCV) RNA, which is associated with viral translation stimulation. By systematic biophysical investigations, we have found that La binds to domain IV using an RNA recognition that is quite distinct from its mode of binding to RNAs with a 3' UUU(OH) trailer: although the La motif and first RNA recognition motif (RRM1) are sufficient for high-affinity binding to 3' oligoU, recognition of HCV domain IV requires the La motif and RRM1 to work in concert with the atypical RRM2 which has not previously been shown to have a significant role in RNA binding. This new mode of binding does not appear sequence specific, but recognizes structural features of the RNA, in particular a double-stranded stem flanked by single-stranded extensions. These findings pave the way for a better understanding of the role of La in viral translation initiation.     

Two‐polymerase mechanisms dictate error‐free and error‐prone translesion DNA synthesis in mammals

DNA replication across blocking lesions occurs by translesion DNA synthesis (TLS), involving a multitude of mutagenic DNA polymerases that operate to protect the mammalian genome. Using a quantitative TLS assay, we identified three main classes of TLS in human cells: two rapid and error‐free, and the third slow and error‐prone. A single gene, REV3L, encoding the catalytic subunit of DNA polymerase ζ (polζ), was found to have a pivotal role in TLS, being involved in TLS across all lesions examined, except for a TT cyclobutane dimer. Genetic epistasis siRNA analysis indicated that discrete two‐polymerase combinations with polζ dictate error‐prone or error‐free TLS across the same lesion. These results highlight the central role of polζ in both error‐prone and error‐free TLS in mammalian cells, and show that bypass of a single lesion may involve at least three different DNA polymerases, operating in different two‐polymerase combinations.     

Protein–protein alternative binding modes do not overlap

Proteins often bind other proteins in more than one way. Thus alternative binding modes is an essential feature of protein interactions. Such binding modes may be detected by X‐ray crystallography and thus reflected in Protein Data Bank. The alternative binding is often observed not for the protein itself but for its structural homolog. The results of this study based on the analysis of a comprehensive set of co‐crystallized protein–protein complexes show that the alternative binding modes generally do not overlap, but are spatially separated. This effect is based on molecular recognition characteristics of the protein structures. The results are also in excellent agreement with the intermolecular energy funnel size estimates obtained previously by an independent methodology. The results provide an important insight into the principles of protein association, as well as potential guidelines for modeling of protein complexes and the design of protein interfaces.     

HIV‐1 IN alternative molecular recognition of DNA induced by raltegravir resistance mutations

Virologic failure during treatment with raltegravir, the first effective drug targeting HIV integrase, is associated with two exclusive pathways involving either Q148H/R/K, G140S/A or N155H mutations. We carried out a detailed analysis of the molecular and structural effects of these mutations. We observed no topological change in the integrase core domain, with conservation of a newly identified Ω‐shaped hairpin containing the Q148 residue, in particular. In contrast, the mutations greatly altered the specificity of DNA recognition by integrase. The native residues displayed a clear preference for adenine, whereas the mutant residues strongly favored pyrimidines. Raltegravir may bind to N155 and/or Q148 residues as an adenine bioisoster. This may account for the selected mutations impairing raltegravir binding while allowing alternative DNA recognition by integrase. This study opens up new opportunities for the design of integrase inhibitors active against raltegravir‐resistant viruses. Copyright © 2009 John Wiley & Sons, Ltd.     

Two modes of Cue2-mediated mRNA cleavage with distinct substrate recognition initiate no-go decay

Ribosome collisions are recognized by E3 ubiquitin ligase Hel2/ZNF598, leading to RQC (ribosome-associated quality control) and to endonucleolytic cleavage and degradation of the mRNA termed NGD (no-go decay). NGD in yeast requires the Cue2 endonuclease and occurs in two modes, either coupled to RQC (NGD^RQC+) or RQC uncoupled (NGD^RQC−). This is mediated by an unknown mechanism of substrate recognition by Cue2. Here, we show that the ubiquitin binding activity of Cue2 is required for NGD^RQC− but not for NGD^RQC+, and that it involves the first two N-terminal Cue domains. In contrast, Trp122 of Cue2 is crucial for NGD^RQC+. Moreover, Mbf1 is required for quality controls by preventing +1 ribosome frameshifting induced by a rare codon staller. We propose that in Cue2-dependent cleavage upstream of the collided ribosomes (NGD^RQC−), polyubiquitination of eS7 is recognized by two N-terminal Cue domains of Cue2. In contrast, for the cleavage within collided ribosomes (NGD^RQC+), the UBA domain, Trp122 and the interaction between Mbf1 and uS3 are critical.     

RNA‐based fluorescent biosensors for live cell detection of bacterial sRNA

Bacteria contain a diverse set of RNAs to provide tight regulation of gene expression in response to environmental stimuli. Bacterial small RNAs (sRNAs) work in conjunction with protein cofactors to bind complementary mRNA sequences in the cell, leading to up‐ or downregulation of protein synthesis. In vivo imaging of sRNAs can aid in understanding their spatiotemporal dynamics in real time, which inspires new ways to manipulate these systems for a variety of applications including synthetic biology and therapeutics. Current methods for sRNA imaging are quite limited in vivo and do not provide real‐time information about fluctuations in sRNA levels. Herein, we describe our efforts toward the development of an RNA‐based fluorescent biosensor for bacterial sRNA both in vitro and in vivo. We validated these sensors for three different bacterial sRNAs in Escherichia coli and demonstrated that the designs provide a bright, sequence‐specific signal output in response to exogenous and endogenous RNA targets.     

Identification of an intronic splicing regulatory element involved in auto‐regulation of alternative splicing of SCL33 pre‐mRNA

In Arabidopsis, pre‐mRNAs of serine/arginine‐rich (SR) proteins undergo extensive alternative splicing (AS). However, little is known about the cis‐elements and trans‐acting proteins involved in regulating AS. Using a splicing reporter (GFP–intron–GFP), consisting of the GFP coding sequence interrupted by an alternatively spliced intron of SCL33, we investigated whether cis‐elements within this intron are sufficient for AS, and which SR proteins are necessary for regulated AS. Expression of the splicing reporter in protoplasts faithfully produced all splice variants from the intron, suggesting that cis‐elements required for AS reside within the intron. To determine which SR proteins are responsible for AS, the splicing pattern of the GFP–intron–GFP reporter was investigated in protoplasts of three single and three double mutants of SR genes. These analyses revealed that SCL33 and a closely related paralog, SCL30a, are functionally redundant in generating specific splice variants from this intron. Furthermore, SCL33 protein bound to a conserved sequence in this intron, indicating auto‐regulation of AS. Mutations in four GAAG repeats within the conserved region impaired generation of the same splice variants that are affected in the scl33 scl30a double mutant. In conclusion, we have identified the first intronic cis‐element involved in AS of a plant SR gene, and elucidated a mechanism for auto‐regulation of AS of this intron.