Validating the EMCV IRES Secondary Structure with Structure-Function Analysis.

The encephalomyocarditis virus internal ribosome entry site (EMCV IRES) is a structured RNA sequence found in the 5' UTR of the genomic RNA of the encephalomyocarditis virus. The EMCV IRES structure facilitates efficient translation initiation without needing a 5' m7G cap or the cap-binding protein eIF4E. The secondary structure of IRES has been the subject of several previous studies, and a number of different structural models have been proposed. Though some domains of the IRES are conserved across the different secondary structure models, domain I of the IRES varies greatly across them. A literature comparison led to the identification of three regions of interest that display structural heterogeneity within past secondary structure models. To test the accuracy of the secondary structure models in these regions, we employed mutational analysis and SHAPE probing. Mutational analysis revealed that two helical regions within the identified regions of interest are important for IRES translation. These helical regions are consistent with only one of the structure predictions in the literature and do not form in EMCV IRES structures predicted using modern secondary structure prediction methods. The importance of these regions is further supported by multiple SHAPE protections when probing was performed after in vitro translation, indicating that these regions are involved in the IRES translation complex. This work validates a published structure and demonstrates the importance of domain I during EMCV IRES translation initiation.

Interaction of the Influenza A Virus NS1 Protein with the 5'-m7G-mRNA·eIF4E·eIF4G1 Complex.

The influenza A virus (IAV) is responsible for seasonal epidemics that result in hundreds of thousands of deaths worldwide annually. The non-structural protein 1 (NS1) of the IAV inflicts various antagonistic processes on the host during infection. These processes include inhibition of the host interferon system, inhibition of the apoptotic response, and enhancement of viral mRNA translation, all of which contribute to the overall virulence of the IAV. Although the mechanism by which NS1 stimulates translation is unknown, NS1 has been shown to bind both poly-A binding Protein 1 and eukaryotic initiation factor 4 gamma 1 (eIF4G1), two proteins necessary for cap-dependent translation. We directly analyzed the interaction between NS1 and eIF4G1 within the context of the 5'-m7G-mRNA·eIF4E·eIF4G1 complex. Interestingly, our studies show that NS1 can bind this complex in the presence or absence of 5'-m7G-mRNA. Additionally, we were interested in investigating whether NS1 interacts with eIF4E directly. Our results indicate that NS1 can bind to eIF4E only in the absence of 5'-m7G-mRNA. Considering previous data, we propose that NS1 stimulates translation by binding to eIF4G1 and recruiting the 43S pre-translation initiation complex to the mRNA.

The Fragile X Protein Disordered Regions Bind a Novel RNA Target.

The fragile X proteins (FXPs) are a family of RNA-binding proteins that regulate mRNA translation to promote proper neural development and cognition in mammals. Of particular interest to researchers is the fragile X mental retardation protein (FMRP), as its absence leads to a neurodevelopmental disorder: fragile X syndrome (FXS), the leading monogenetic cause of autism spectrum disorders. A primary focus of research has been to determine mRNA targets of the FXPs in vivo through pull-down techniques, and to validate them through in vitro binding studies; another approach has been to perform in vitro selection experiments to identify RNA sequence and structural targets. These mRNA targets can be further investigated as potential targets for FXS therapeutics. The most established RNA structural target of this family of proteins is the G-quadruplex. In this article, we report a 99 nucleotide RNA target that is bound by all three FXPs with nanomolar equilibrium constants. Furthermore, we determined that the last 102 amino acids of FMRP, which includes the RGG motif, were necessary and sufficient to bind this RNA target. To the best of our knowledge, this is one of only a few examples of non-G-quadruplex, non-homopolymer RNAs bound by the RGG motif/C-termini of FMRP.

PABP1 Drives the Selective Translation of Influenza A Virus mRNA.

Influenza A virus (IAV) is a human-infecting pathogen with a history of causing seasonal epidemics and on several occasions worldwide pandemics. Infection by IAV causes a dramatic decrease in host mRNA translation, whereas viral mRNAs are efficiently translated. The IAV mRNAs have a highly conserved 5'-untranslated region (5'UTR) that is rich in adenosine residues. We show that the human polyadenylate binding protein 1 (PABP1) binds to the 5'UTR of the viral mRNAs. The interaction of PABP1 with the viral 5'UTR makes the translation of viral mRNAs more resistant to canonical cap-dependent translation inhibition than model mRNAs. Additionally, PABP1 bound to the viral 5'UTR can recruit eIF4G in an eIF4E-independent manner. These results indicate that PABP1 bound to the viral 5'UTR may promote eIF4E-independent translation initiation.

The Fragile X Proteins Differentially Regulate Translation of Reporter mRNAs with G-quadruplex Structures.

Fragile X Syndrome, as well as some manifestations of autism spectrum disorder, results from improper RNA regulation due to a deficiency of fragile X mental retardation protein (FMRP). FMRP and its autosomal paralogs, fragile X related proteins 1 & 2 (FXR1P/2P), have been implicated in many aspects of RNA regulation, from protein synthesis to mRNA stability and decay. The literature on the fragile X related proteins' (FXPs) role in mRNA regulation and their potential mRNA targets is vast. Therefore, we developed an approach to investigate the function of FXPs in translational control using three potential mRNA targets. Briefly, we first selected top mRNA candidates found to be associated with the FXPs and whose translation are influenced by one or more of the FXPs. We then narrowed down the FXPs' binding site(s) within the mRNA, analyzed the strength of this binding in vitro, and determined how each FXP affects the translation of a minimal reporter mRNA with the binding site. Overall, all FXPs bound with high affinity to RNAs containing G-quadruplexes, such as Cyclin Dependent Kinase Inhibitor p21 and FMRP's own coding region. Interestingly, FMRP inhibited the translation of each mRNA distinctly and in a manner that appears to correlate with its binding to each mRNA. In contrast, FXR1P/2P inhibited all mRNAs tested. Finally, although binding of our RNAs was due to the RGG (arginine-glycine-glycine) motif-containing C-terminal region of the FXPs, this region was not sufficient to cause inhibition of translation.

Controlling Protein Enrichment in Lipid Sponge Phase Droplets using SNAP-Tag Bioconjugation.

All cells use organized lipid compartments to facilitate specific biological functions. Membrane-bound organelles create defined spatial environments that favor unique chemical reactions while isolating incompatible biological processes. Despite the fundamental role of cellular organelles, there is a scarcity of methods for preparing functional artificial lipid-based compartments. Here, we demonstrate a robust bioconjugation system for sequestering proteins into zwitterionic lipid sponge phase droplets. Incorporation of benzylguanine (BG)-modified phospholipids that form stable covalent linkages with an O6 -methylguanine DNA methyltransferase (SNAP-tag) fusion protein enables programmable control of protein capture. We show that this methodology can be used to anchor hydrophilic proteins at the lipid-aqueous interface, concentrating them within an accessible but protected chemical environment. SNAP-tag technology enables the integration of proteins that regulate complex biological functions in lipid sponge phase droplets, and should facilitate the development of advanced lipid-based artificial organelles.

Discovery of a pre-mRNA structural scaffold as a contributor to the mammalian splicing code.

The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of 3D structural scaffold of AdML-a model pre-mRNA substrate-guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA. We further find that the pre-mRNA segments potentially interacting with the early spliceosomal component U1 snRNP are distributed across the intron, that there is a spatial proximity of 5' and 3' splice sites within the pre-mRNA scaffold, and that an interplay exists between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components can recognize a 3D structural scaffold beyond the short splice signal sequences, and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This provides a conceptual basis to analyze the contribution of recognizable 3D structural scaffolds to the splicing code across the mammalian transcriptome.

Influenza A Virus NS1 Protein Binds as a Dimer to RNA-Free PABP1 but Not to the PABP1·Poly(A) RNA Complex.

Influenza A virus (IAV) is a highly contagious human pathogen that is responsible for tens of thousands of deaths each year. Non-structural protein 1 (NS1) is a crucial protein expressed by IAV to evade the host immune system. Additionally, NS1 has been proposed to stimulate translation because of its ability to bind poly(A) binding protein 1 (PABP1) and eukaryotic initiation factor 4G. We analyzed the interaction of NS1 with PABP1 using quantitative techniques. Our studies show that NS1 binds as a homodimer to PABP1, and this interaction is conserved across different IAV strains. Unexpectedly, NS1 does not bind to PABP1 that is bound to poly(A) RNA. Instead, NS1 binds only to PABP1 free of RNA, suggesting that stimulation of translation does not occur by NS1 interacting with the PABP1 molecule attached to the mRNA 3'-poly(A) tail. These results suggest that the function of the NS1·PABP1 complex appears to be distinct from the classical role of PABP1 in translation initiation, when it is bound to the 3'-poly(A) tail of mRNA.

The Human Fragile X Mental Retardation Protein Inhibits the Elongation Step of Translation through Its RGG and C-Terminal Domains.

The fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates the translation of numerous mRNAs in neurons. The precise mechanism of translational regulation by FMRP is unknown. Some studies have indicated that FMRP inhibits the initiation step of translation, whereas other studies have indicated that the elongation step of translation is inhibited by FMRP. To determine whether FMRP inhibits the initiation or the elongation step of protein synthesis, we investigated m7G-cap-dependent and IRES-driven, cap-independent translation of several reporter mRNAs in vitro. Our results show that FMRP inhibits both m7G-cap-dependent and cap-independent translation to similar degrees, indicating that the elongation step of translation is inhibited by FMRP. Additionally, we dissected the RNA-binding domains of hFMRP to determine the essential domains for inhibiting translation. We show that the RGG domain, together with the C-terminal domain (CTD), is sufficient to inhibit translation, while the KH domains do not inhibit mRNA translation. However, the region between the RGG domain and the KH2 domain may contribute as NT-hFMRP shows more potent inhibition than the RGG-CTD tail alone. Interestingly, we see a correlation between ribosome binding and translation inhibition, suggesting the RGG-CTD tail of hFMRP may anchor FMRP to the ribosome during translation inhibition.

A simple procedure for bacterial expression and purification of the fragile X protein family.

The fragile X protein family consists of three RNA-binding proteins involved in translational regulation. Fragile X mental retardation protein (FMRP) is well-studied, as its loss leads to fragile X syndrome, a neurodevelopmental disorder which is the most prevalent form of inherited mental retardation and the primary monogenetic cause of autism. Fragile X related proteins 1 and 2 (FXR1P and FXR2P) are autosomal paralogs of FMRP that are involved in promoting muscle development and neural development, respectively. There is great interest in studying this family of proteins, yet researchers have faced much difficulty in expressing and purifying the full-length versions of these proteins in sufficient quantities. We have developed a simple, rapid, and inexpensive procedure that allows for the recombinant expression and purification of full-length human FMRP, FXR1P, and FXR2P from Escherichia coli in high yields, free of protein and nucleic acid contamination. In order to assess the proteins' function after purification, we confirmed their binding to pseudoknot and G-quadruplex forming RNAs as well as their ability to regulate translation in vitro.

RNA-Binding Specificity of the Human Fragile X Mental Retardation Protein.

Fragile X syndrome is the most common form of inherited intellectual disability and is caused by a deficiency of the fragile X mental retardation protein (FMRP) in neurons. FMRP regulates the translation of numerous mRNAs within dendritic synapses, but how FMRP recognizes these target mRNAs remains unknown. FMRP has KH0, KH1, KH2, and RGG domains, which are thought to bind to specific RNA recognition elements (RREs). Several studies used high-throughput methods to identify various RREs in mRNAs that FMRP may bind to in vivo. However, there is little overlap in the mRNA targets identified by each study, suggesting that the RNA-binding specificity of FMRP is still unknown. To determine the specificity of FMRP for the RREs, we performed quantitative in vitroRNA binding studies with various constructs of human FMRP. Unexpectedly, our studies show that the KH domains do not bind to the previously identified RREs. To further investigate the RNA-binding specificity of FMRP, we developed a new method called Motif Identification by Analysis of Simple sequences (MIDAS) to identify single-stranded RNA sequences bound by KH domains. We find that the FMRP KH0, KH1, and KH2 domains bind weakly to the single-stranded RNA sequences suggesting that they may have evolved to bind more complex RNA structures. Additionally, we find that the RGG motif of human FMRP binds with a high affinity to an RNAG-quadruplex structure that lacks single-stranded loops, double-stranded stems, or junctions.

Translation of non-standard codon nucleotides reveals minimal requirements for codon-anticodon interactions.

The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N1 of purines and the N3 of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position. Inosine, found in eukaryotic mRNAs, is an important example of destabilization of the codon-anticodon interaction. Whereas single inosines are efficiently translated, multiple inosines, e.g., in the serotonin receptor 5-HT2C mRNA, inhibit translation. Thus, our results indicate that despite the robustness of the decoding process, its tolerance toward the weakening of codon-anticodon interactions is limited.

RNA Modulates the Interaction between Influenza A Virus NS1 and Human PABP1.

Nonstructural protein 1 (NS1) is a multifunctional protein involved in preventing host-interferon response in influenza A virus (IAV). Previous studies have indicated that NS1 also stimulates the translation of viral mRNA by binding to conserved sequences in the viral 5'-UTR. Additionally, NS1 binds to poly(A) binding protein 1 (PABP1) and eukaryotic initiation factor 4G (eIF4G). The interaction of NS1 with the viral 5'-UTR, PABP1, and eIF4G has been suggested to specifically enhance the translation of viral mRNAs. In contrast, we report that NS1 does not directly bind to sequences in the viral 5'-UTR, indicating that NS1 is not responsible for providing the specificity to stimulate viral mRNA translation. We also monitored the interaction of NS1 with PABP1 using a new, quantitative FRET assay. Our data show that NS1 binds to PABP1 with high affinity; however, the binding of double-stranded RNA (dsRNA) to NS1 weakens the binding of NS1 to PABP1. Correspondingly, the binding of PABP1 to NS1 weakens the binding of NS1 to double-stranded RNA (dsRNA). In contrast, the affinity of PABP1 for binding to poly(A) RNA is not significantly changed by NS1. We propose that the modulation of NS1·PABP1 interaction by dsRNA may be important for the viral cycle.

Optimization of ClpXP activity and protein synthesis in an E. coli extract-based cell-free expression system.

Protein degradation is a fundamental process in all living cells and is essential to remove both damaged proteins and intact proteins that are no longer needed by the cell. We are interested in creating synthetic genetic circuits that function in a cell-free expression system. This will require not only an efficient protein expression platform but also a robust protein degradation system in cell extract. Therefore, we purified and tested the activity of E. coli ClpXP protease in cell-free transcription-translation (TX-TL) systems that used E. coli S30 cell extract. Surprisingly, our studies showed that purified ClpXP added to the TX-TL system has very low proteolytic activity. The low activity of ClpXP was correlated with the rapid consumption of adenosine triphosphate (ATP) in cell extract. We improved the activity of ClpXP in cell extract by adding exogenous ATP and an energy regeneration system. We then established conditions for both protein synthesis, and protein degradation by ClpXP to occur simultaneously in the TX-TL systems. The optimized conditions for ClpXP activity will be useful for creating tunable synthetic genetic circuits and in vitro synthetic biology.

Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release.

Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons.

Determination of nucleoside triphosphatase activities from measurement of true inorganic phosphate in the presence of labile phosphate compounds.

One of the most common assays for nucleoside triphosphatase (NTPase) activity entails the quantification of inorganic phosphate (Pi) as a colored phosphomolybdate complex at low pH. While this assay is very sensitive, it is not selective for Pi in the presence of labile organic phosphate compounds (OPCs). Since NTPase activity assays typically require a large excess of OPCs, such as nucleotides, selectivity for Pi in the presence of OPCs is often critical in evaluating enzyme activity. Here we present an improved method for the measurement of enzymatic nucleotide hydrolysis as Pi released, which achieves selectivity for Pi in the presence of OPCs while also avoiding the costs and hazards inherent in other methods for measuring nucleotide hydrolysis. We apply this method to the measurement of ATP hydrolysis by nitrogenase and GTP hydrolysis by elongation factor G (EF-G) in order to demonstrate the broad applicability of our method for the determination of nucleotide hydrolysis in the presence of interfering OPCs.

Mechanism of Translation Termination: RF1 Dissociation Follows Dissociation of RF3 from the Ribosome.

Release factors 1 and 2 (RF1 and RF2, respectively) bind to ribosomes that have a stop codon in the A site and catalyze the release of the newly synthesized protein. Following peptide release, the dissociation of RF1 and RF2 from the ribosome is accelerated by release factor 3 (RF3). The mechanism for RF3-promoted dissociation of RF1 and RF2 is unclear. It was previously proposed that RF3 hydrolyzes GTP and dissociates from the ribosome after RF1 dissociation. Here we monitored directly the dissociation kinetics of RF1 and RF3 using Förster resonance energy transfer-based assays. In contrast to the previous model, our data show that RF3 hydrolyzes GTP and dissociates from the ribosome before RF1 dissociation. We propose that RF3 stabilizes the ratcheted state of the ribosome, which consequently accelerates the dissociation of RF1 and RF2.

Ribosome Induces a Closed to Open Conformational Change in Release Factor 1.

Bacterial translation termination is triggered when a stop codon arrives at the ribosomal A site. Stop codons are recognized by class I release factors (RF1 and RF2 in Escherichia coli), which bind to the ribosome and catalyze the release of the newly synthesized protein. Crystal structures showed that RF1 and RF2 are in an open conformation when bound to the ribosome but are in a closed conformation when not bound to the ribosome. It is not clear whether only the open form of RF1 and RF2 binds to the ribosome. Alternatively, the closed form of RF1 and RF2 may bind to the ribosome and undergo a conformational change to the open state upon binding. We used transition metal ion fluorescence resonance energy transfer experiments to monitor precisely the conformation of RF1 in the absence and presence of the ribosome. Our results indicate that RF1 undergoes a large conformational change from a closed to an open form upon binding to the ribosome. Our results are consistent with the mechanism, in which high termination fidelity is achieved by linking stop codon recognition by RF1 to the change in conformation from closed to open state.

Fragile X mental retardation protein: A paradigm for translational control by RNA-binding proteins.

Translational control is a common mechanism used to regulate gene expression and occur in bacteria to mammals. Typically in translational control, an RNA-binding protein binds to a unique sequence in the mRNA to regulate protein synthesis by the ribosomes. Alternatively, a protein may bind to or modify a translation factor to globally regulate protein synthesis by the cell. Here, we review translational control by the fragile X mental retardation protein (FMRP), the absence of which causes the neurological disease, fragile X syndrome (FXS).