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Results for “RNA tertiary structure”
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Alternate, virus-induced membrane rearrangements support positive-strand RNA virus genome replication.
All positive-strand RNA [(+)RNA] viruses replicate their RNA on intracellular membranes, often in association with spherular invaginations of the target membrane. For brome mosaic virus, we previously showed that such spherules serve as compartments or mini-organelles for RNA replication and that their assembly, structure, and function have similarities to the replicative cores of retrovirus and double-stranded RNA virus virions. Some other (+)RNA viruses conduct RNA replication in association with individual or clustered double-membrane vesicles, appressed double membranes, or other structures whose possible relationships to the spherular invaginations are unclear. Here we show that modulating the relative levels and interactions of brome mosaic virus replication factors 1a and 2a polymerase (2apol) shifted the membrane rearrangements associated with RNA replication from small invaginated spherules to large, karmellae-like, multilayer stacks of appressed double membranes that supported RNA replication as efficiently as spherules. Spherules were induced by expressing 1a, which has functional similarities to retrovirus virion protein Gag, or 1a plus low levels of 2apol. Double-membrane layers were induced by 1a plus higher levels of 2apol and were suppressed by deleting the major 1a-interacting domain from 2apol. The stacked, double-membrane layers alternated with spaces that, like spherule interiors, were 50-60 nm wide, connected to the cytoplasm, and contained 1a and 2apol. These and other results suggest that seemingly diverse membrane rearrangements associated with RNA replication by varied (+)RNA viruses may represent topologically and functionally related structures formed by similar protein-protein and protein-membrane interactions and interconverted by altering the balances among those interactions.
Determinants of Rbp1p localization in specific cytoplasmic mRNA-processing foci, P-bodies.
Rbp1p, a yeast RNA-binding protein, decreases the level of mitochondrial porin mRNA by enhancing its degradation, but the intracellular location of the Rbp1p-mediated degradation complex remains unknown. We show here that Rbp1p in xrn1Delta mutant yeast localizes in specific cytoplasmic foci that are known as P-bodies. The N-terminal and RNA recognition motif (RRM) 1 domains of Rbp1p are necessary but not sufficient for its localization in P bodies. Rbp1p forms oligomers through its C-terminal domain in vivo; N-terminal-delete, or RRM1-mutated Rbp1p can be more efficiently recruited to P-bodies in an xrn1Delta strain, expressing a full-length Rbp1p. Although POR1 mRNA is localized to P bodies in an xrn1Delta strain, this localization does not depend on Rbp1p. Decapping activator Dhh1p directly interacts with Rbp1p. However, the recruitment of Rbp1p to P-bodies does not require Dhh1p or Ccr4p. In wild-type cells, Rbp1p can localize to P-bodies under glucose deprivation or treatment with KCl. In addition, Rbp1p-mediated porin mRNA decay is elicited by Xrn1p, a 5 ' to 3 ' exonuclease. These results provide new insight into the mechanism of Rbp1p function.
Functional divergence of a unique C-terminal domain of leucyl-tRNA synthetase to accommodate its splicing and aminoacylation roles.
Leucyl-tRNA synthetase (LeuRS) performs dual essential roles in group I intron RNA splicing as well as protein synthesis within the yeast mitochondria. Deletions of the C terminus differentially impact the two functions of the enzyme in splicing and aminoacylation in vivo. Herein, we determined that a fiveamino acid C-terminal deletion of LeuRS, which does not complement a null strain, can form a ternary complex with the bI4 intron and its maturase splicing partner. However, the complex fails to stimulate splicing activity. The x-ray co-crystal structure of LeuRS showed that a C-terminal extension of about 60 amino acids forms a discrete domain, which is unique among the LeuRSs and interacts with the corner of the L-shaped tRNALeu. Interestingly, deletion of the entire yeast mitochondrial LeuRS C-terminal domain enhanced its aminoacylation and amino acid editing activities. In striking contrast, deletion of the corresponding C-terminal domain of Escherichia coli LeuRS abolished aminoacylation of tRNALeu and also amino acid editing of mischarged tRNA molecules. These results suggest that the role of the leucine-specific C-terminal domain in tRNA recognition for aminoacylation and amino acid editing has adapted differentially and with surprisingly opposite effects. We propose that the secondary role of yeast mitochondrial LeuRS in RNA splicing has impacted the functional evolution of this critical C-terminal domain.
Crystal structure of Ski8p, a WD-repeat protein with dual roles in mRNA metabolism and meiotic recombination.
Ski8p is a WD-repeat protein with an essential role for the Ski complex assembly in an exosome-dependent 3'-to-5' mRNA decay. In addition, Ski8p is involved in meiotic recombination by interacting with Spo11p protein. We have determined the crystal structure of Ski8p from Saccharomyces cerevisiae at 2.2 A resolution. The structure reveals that Ski8p folds into a seven-bladed beta propeller. Mapping sequence conservation and hydrophobicities of amino acids on the molecular surface of Ski8p reveals a prominent site on the top surface of the beta propeller, which is most likely involved in mediating interactions of Ski8p with Ski3p and Spo11p. Mutagenesis combined with yeast two-hybrid and GST pull-down assays identified the top surface of the beta propeller as being required for Ski8p binding to Ski3p and Spo11p. The functional implications for Ski8p function in both mRNA decay and meiotic recombination are discussed.
Physical and functional interaction between the eukaryotic orthologs of prokaryotic translation initiation factors IF1 and IF2.
To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site.
Sox17 and beta-catenin cooperate to regulate the transcription of endodermal genes.
Recent studies have led to a model of the molecular pathway that specifies the endoderm during vertebrate gastrulation. The HMG box transcription factor Sox17 is a key component of this pathway and is essential for endoderm formation; however, the molecular events controlled by Sox17 are largely unknown. We have identified several direct transcriptional targets of Sox17, including Foxa1 and Foxa2. We show that beta-catenin, a component of Wnt signaling pathway, physically interacts with Sox17 and potentiates its transcriptional activation of target genes. We identify a motif in the C terminus of Sox17, which is conserved in all the SoxF subfamily of Sox proteins, and this motif is required for the ability of Sox17 to both transactivate target genes and bind beta-catenin. Nuclear beta-catenin is present in endoderm cells of the gastrula, and depletion of beta-catenin from embryos results in a repression of Sox17 target genes. These data suggest that in a mechanism analogous to Tcf/Lef interacting with beta-catenin, Sox17 and beta-catenin interact to transcribe endodermal target genes.
RS domains contact the pre-mRNA throughout spliceosome assembly.
SR proteins are essential metazoan splicing factors that contain an RNA-binding domain and an arginine/serine-rich domain that functions to promote assembly of the spliceosome. The prevailing model over the past several years suggests that the RS domains function as protein-interaction domains. However, two new papers from Green et al. demonstrate that these RS domains directly contact the pre-mRNA within the functional spliceosome. The sequential character of these contacts suggests that RS domain interactions with RNA promote spliceosome assembly.
Wrong PH for RNA degradation.
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Rift Valley fever virus L segment: correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerases.
The sequence of Rift Valley fever virus L segment that we published in a previous paper was erroneous in the 3'-terminal region of the antigenomic RNA molecule. Here, we have shown that the L segment is in fact 6404 nucleotides long and encodes a polypeptide of 237.7K in the viral complementary sense. Sequence comparisons performed between the RNA-dependent RNA polymerases of 22 negative-stranded RNA viruses revealed the existence of two novel regions located at the amino termini of the proteins and conserved only in the polymerases of bunya- and arenaviruses. In the region conserved in all RNA-dependent polymerases, corresponding to the so-called 'polymerase module', we identified a new motif, designated premotif A, common to all RNA-dependent polymerases, as well as amino acids located in the region between motifs preA and A which are strictly conserved for segmented negative-stranded RNA viruses. Using the recently released coordinates of human immunodeficiency virus reverse transcriptase and the alignment between all RNA-dependent polymerases in the 'polymerase module', we have determined the position of the conserved residues in these polymerases and discuss their possible functions in light of the available structural information.
Formation of higher-order secondary and tertiary chromatin structures by genomic mouse mammary tumor virus promoters.
Agarose multigel electrophoresis has been used to characterize the structural features of isolated genomic mouse mammary tumor virus (MMTV) promoters. The mouse 3134 cells used for these studies contain approximately 200 stably integrated tandem repeats of a 2.4-kb MMTV promoter fragment. Inactive, basally active, and hormonally activated genomic promoters were liberated by restriction digestion of isolated nuclei, recovered in low-salt nuclear extracts, and electrophoresed in multigels consisting of nine individual agarose running gels. Specific bands were detected and characterized by Southern and Western blotting. We find that transcriptionally inactive promoters contain TBP and high levels of histone H1, and are present to varying extents in both untreated and dexamethasone (DEX)-treated 3134 cells. In contrast, the basally active promoter, present in untreated cells, is bound to RNA Pol II, TBP, and Oct1, contains acetylated H3 tail domains, and is depleted of histone H1. The DEX-activated promoter possessed similar composition as the basal promoter, but also contains stably bound Brg1. Strikingly, all forms of the MMTV promoter condense into higher-order secondary and/or tertiary chromatin structures in vitro in the presence of Mg2+. Thus, genomic MMTV promoter chromatin retains the ability to form classical higher-order structures under physiological salt conditions, even after dissociation of H1 and binding of several transcription factors and multiprotein complexes. These results suggest that transcriptionally active eukaryotic promoters may function in a locally folded chromatin environment in vivo.
Dissecting nucleases into their structural and functional domains. Mapping the RNA-binding surface of RNase III by NMR.
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The V proteins of simian virus 5 and other paramyxoviruses inhibit induction of interferon-beta.
In this article we show that the paramyxovirus SV5 is a poor inducer of interferon-beta (IFN-beta). This inefficient induction is a consequence of the expression of an intact viral V protein. In the absence of the viral V protein cysteine-rich C-terminal domain, IFN-beta mRNA is strongly induced and the transcription factors NF-kappaB and IRF-3 are activated significantly. The V protein can work in isolation from SV5 to block intracellular dsRNA signaling. The mechanism of block to dsRNA signaling is distinct from that previously observed for blocking IFN signaling in that proteolysis of candidate factors cannot be detected, and furthermore, the respective blocks require distinct protein domains. Blocking of the induction of IFN-beta by dsRNA requires the C-terminal cysteine-rich domain, a feature that is highly conserved among paramyxoviruses. We demonstrate that the V proteins from other paramyxoviruses have equivalent functions and speculate that limiting the yield of IFN-beta during infection may be a general property of paramyxoviruses.
Crystal structure of UAP56, a DExD/H-box protein involved in pre-mRNA splicing and mRNA export.
UAP56 is an essential eukaryotic pre-mRNA splicing factor and mRNA export factor. The mechanisms of its functions are not well understood. We determined the crystal structures of the N- and C-terminal domains of human UAP56 (comprising 90% of the full-length UAP56) at 1.9 A resolution. The two domains each have a RecA-like fold and are connected by a flexible linker. The overall fold of each domain is highly similar to the corresponding domains of eIF4A (a prototypic DExD/H-box protein), with differences at the loops and termini. This structural similarity suggests that UAP56 is likely to possess ATPase and helicase activity similar to eIF4A. The NTP binding pocket of UAP56 is occupied by a citrate ion, mimicking the phosphates of NTP and retaining the P loop in an open conformation. The crystal structure of the N-terminal domain of UAP56 also reveals a dimer interface that is potentially important for UAP56's function.
Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins.
mRNA 5'-cap recognition by the eukaryotic translation initiation factor eIF4E has been exhaustively characterized with the aid of a novel fluorometric, time-synchronized titration method, and X-ray crystallography. The association constant values of recombinant eIF4E for 20 different cap analogues cover six orders of magnitude; with the highest affinity observed for m(7)GTP (approximately 1.1 x 10(8) M(-1)). The affinity of the cap analogues for eIF4E correlates with their ability to inhibit in vitro translation. The association constants yield contributions of non-covalent interactions involving single structural elements of the cap to the free energy of binding, giving a reliable starting point to rational drug design. The free energy of 7-methylguanine stacking and hydrogen bonding (-4.9 kcal/mol) is separate from the energies of phosphate chain interactions (-3.0, -1.9, -0.9 kcal/mol for alpha, beta, gamma phosphates, respectively), supporting two-step mechanism of the binding. The negatively charged phosphate groups of the cap act as a molecular anchor, enabling further formation of the intermolecular contacts within the cap-binding slot. Stabilization of the stacked Trp102/m(7)G/Trp56 configuration is a precondition to form three hydrogen bonds with Glu103 and Trp102. Electrostatically steered eIF4E-cap association is accompanied by additional hydration of the complex by approximately 65 water molecules, and by ionic equilibria shift. Temperature dependence reveals the enthalpy-driven and entropy-opposed character of the m(7)GTP-eIF4E binding, which results from dominant charge-related interactions (DeltaH degrees =-17.8 kcal/mol, DeltaS degrees= -23.6 cal/mol K). For recruitment of synthetic eIF4GI, eIF4GII, and 4E-BP1 peptides to eIF4E, all the association constants were approximately 10(7) M(-1), in decreasing order: eIF4GI>4E-BP1>eIF4GII approximately 4E-BP1(P-Ser65) approximately 4E-BP1(P-Ser65/Thr70). Phosphorylation of 4E-BP1 at Ser65 and Thr70 is insufficient to prevent binding to eIF4E. Enhancement of the eIF4E affinity for cap occurs after binding to eIF4G peptides.
Regulation of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2.
RIG-I is an RNA helicase containing caspase activation and recruitment domains (CARDs). RNA binding and signaling by RIG-I are implicated in pathogen recognition and triggering of IFN-alpha/beta immune defenses that impact cell permissiveness for hepatitis C virus (HCV). Here we evaluated the processes that control RIG-I signaling. RNA binding studies and analysis of cells lacking RIG-I, or the related MDA5 protein, demonstrated that RIG-I, but not MDA5, efficiently binds to secondary structured HCV RNA to confer induction of IFN-beta expression. We also found that LGP2, a helicase related to RIG-I and MDA5 but lacking CARDs and functioning as a negative regulator of host defense, binds HCV RNA. In resting cells, RIG-I is maintained as a monomer in an autoinhibited state, but during virus infection and RNA binding it undergoes a conformation shift that promotes self-association and CARD interactions with the IPS-1 adaptor protein to signal IFN regulatory factor 3- and NF-kappaB-responsive genes. This reaction is governed by an internal repressor domain (RD) that controls RIG-I multimerization and IPS-1 interaction. Deletion of the RIG-I RD resulted in constitutive signaling to the IFN-beta promoter, whereas RD expression alone prevented signaling and increased cellular permissiveness to HCV. We identified an analogous RD within LGP2 that interacts in trans with RIG-I to ablate self-association and signaling. Thus, RIG-I is a cytoplasmic sensor of HCV and is governed by RD interactions that are shared with LGP2 as an on/off switch controlling innate defenses. Modulation of RIG-I/LGP2 interaction dynamics may have therapeutic implications for immune regulation.
ATP requirement for Prp5p function is determined by Cus2p and the structure of U2 small nuclear RNA.
Stable addition of U2 small nuclear ribonucleoprotein (snRNP) to form the prespliceosome is the first ATP-dependent step in splicing, and it requires the DEXD/H box ATPase Prp5p. However, prespliceosome formation occurs without ATP in extracts lacking the U2 snRNP protein Cus2p. Here we show that Prp5p is required for the ATP-independent prespliceosome assembly that occurs in the absence of Cus2p. Addition of recombinant Cus2p can restore the ATP dependence of prespliceosome assembly, but only if it is added before Prp5p. Prp5p with an altered ATP-binding domain (Prp5-GNTp) can support growth in vivo, but only in a cus2 deletion strain, mirroring the in vitro results. Other Prp5 ATP-binding domain substitutions are lethal, even in the cus2 deletion strain, but can be suppressed by U2 small nuclear RNA mutations that hyperstabilize U2 stem IIa. We infer that the presence of Cus2p and stem IIa-destabilized forms of U2 small nuclear RNA places high demands on the ATP-driven function of Prp5p. Because Prp5p is not dispensable in vitro even in the absence of ATP, we propose that the core Prp5p function in bringing U2 to the branchpoint is not directly ATP-dependent. The positive role of Cus2p in rescuing mutant U2 can be reconciled with its antagonistic effect on Prp5 function in a model whereby Cus2p first helps Prp5p to activate the U2 snRNP for prespliceosome formation but then is displaced by Prp5p before or during the stabilization of U2 at the branchpoint.
The NUG1 GTPase reveals and N-terminal RNA-binding domain that is essential for association with 60 S pre-ribosomal particles.
The putative yeast GTPase Nug1, which is associated with several pre-60 S particles in the nucleolus and nucleoplasm, consists of an N-terminal domain, which is found only in eukaryotic orthologues, and middle and C-terminal domains that are conserved throughout eukaryotes, bacteria, and archaea. Here, we analyzed the role of the eukaryote-specific Nug1 N-domain (Nug1-N). We show that the essential Nug1-N is sufficient and necessary for nucle(ol)ar targeting and association with pre-60 S particles. Nug1-N exhibits RNA binding activity and is genetically linked in an allele-specific way to the pre-60 S factors Noc2, Noc3, and Dbp10. In contrast, the middle domain, which exhibits a circularly permuted GTPase fold and an intrinsic GTP hydrolysis activity in vitro, is not essential for cell growth. The conserved Nug1 C-domain, which has a yet uncharacterized fold, is also essential for ribosome biogenesis. Our findings suggest that Nug1 associates with pre-60 S subunits via its essential N-terminal RNA-binding domain and exerts a non-essential regulative role in pre-60 S subunit biogenesis via its central GTPase domain.