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PubMed2004

The kink-turn motif in RNA is dimorphic, and metal ion-dependent.

The kink-turn (K-turn) is a new motif in RNA structure that was identified by examination of the crystal structures of the ribosome. We examined the structural and dynamic properties of this element in free solution. The K-turn RNA exists in a dynamic equilibrium between a tightly kinked conformation and a more open structure similar to a simple bulge bend. The highly kinked form is stabilized by the noncooperative binding of metal ions, but a significant population of the less-kinked form is present even in the presence of relatively high concentrations of divalent metal ions. The conformation of the tightly kinked population is in excellent agreement with that of the K-turn structures observed in the ribosome by crystallography. The end-to-end FRET efficiency of this species agrees closely with that of the ribosomal K-turn, and the direction of the bend measured by comparative gel electrophoresis also corresponds very well. These results show that the tightly kinked conformation of the K-turn requires stabilization by other factors, possibly by protein binding, for example. The K-turn is therefore unlikely to be of itself a primary organizing feature in RNA.

Electrophoresis, Polyacrylamide Gel↗

PubMed2005

A -1 frameshift in the HIV-1 env gene is enhanced by arginine deficiency via a hungry codon mechanism.

Ribosomal frameshifting is used by various organisms to maximize protein coding potential of genomic sequences. It is commonly exploited by RNA viruses to overcome the constraint of their limited genome size. Frameshifting requires specific RNA structural features, such as a suitable heptanucleotide "slippery" sequence and an RNA pseudoknot. Previous genomic analysis of HIV-1 indicated the potential for several hidden genes encoded through frameshifting; one of these, overlapping the envelope gene, has an RNA pseudoknot just downstream from a slippery sequence, AAAAAGA that features an adenine quadruplet prior to a potential hungry arginine codon (AGA). This env-frameshift (env-fs) gene has been shown to encode a truncated glutathione peroxidase homologue, with both antioxidant and anti-apoptotic activities in transfected cells. Using a dual reporter cell-based frameshift assay, we demonstrate that the env-fs frameshift sequence is active in vitro. Furthermore, in arginine deficient media, env-fs frameshifting increased over 100% (p<0.005), consistent with the hypothesized hungry codon mechanism. As a response to arginine deficiency, increased expression of the antioxidant viral GPx gene (env-fs) by upregulation of frameshifting could be protective to HIV-infected cells, as a countermeasure to the increased oxidative stress induced by arginine deficiency (because NO is a known scavenger of hydroxyl radical).

Arginine↗

PubMed2022

ADAR regulates APOL1 via A-to-I RNA editing by inhibition of MDA5 activation in a paradoxical biological circuit.

APOL1 risk variants are associated with increased risk of kidney disease in patients of African ancestry, but not all individuals with the APOL1 high-risk genotype develop kidney disease. As APOL1 gene expression correlates closely with the degree of kidney cell injury in both cell and animal models, the mechanisms regulating APOL1 expression may be critical determinants of risk allele penetrance. The APOL1 messenger RNA includes Alu elements at the 3' untranslated region that can form a double-stranded RNA structure (Alu-dsRNA) susceptible to posttranscriptional adenosine deaminase acting on RNA (ADAR)-mediated adenosine-to-inosine (A-to-I) editing, potentially impacting gene expression. We studied the effects of ADAR expression and A-to-I editing on APOL1 levels in podocytes, human kidney tissue, and a transgenic APOL1 mouse model. In interferon-γ (IFN-γ)-stimulated human podocytes, ADAR down-regulates APOL1 by preventing melanoma differentiation-associated protein 5 (MDA5) recognition of dsRNA and the subsequent type I interferon (IFN-I) response. Knockdown experiments showed that recognition of APOL1 messenger RNA itself is an important contributor to the MDA5-driven IFN-I response. Mathematical modeling suggests that the IFN-ADAR-APOL1 network functions as an incoherent feed-forward loop, a biological circuit capable of generating fast, transient responses to stimuli. Glomeruli from human kidney biopsies exhibited widespread editing of APOL1 Alu-dsRNA, while the transgenic mouse model closely replicated the edited sites in humans. APOL1 expression in mice was inversely correlated with Adar1 expression under IFN-γ stimuli, supporting the idea that ADAR regulates APOL1 levels in vivo. ADAR-mediated A-to-I editing is an important regulator of APOL1 expression that could impact both penetrance and severity of APOL1-associated kidney disease.

Humans↗

PubMed1992

Conformity of RNAs that interact with tetranucleotide loop binding proteins.

A group of RNA binding proteins, termed tetraloop binding proteins, includes ribosomal protein S15 and protein SRP19 of signal recognition particle. They are primary RNA binding proteins, recognize RNA tetranucleotide loops with a GNAR consensus motif, and require a helical region located adjacent to the tetraloop. Closely related RNA structures that fit these criteria appear in helix 6 of SRP RNA, in helices 22 and 23A of 16 S ribosomal RNA, and, as a pseudoknot, in the regulatory region of the rpsO gene.

Amino Acid Sequence↗

PubMed1995

MIRs are classic, tRNA-derived SINEs that amplified before the mammalian radiation.

Short Interspersed Nucleotide Elements (SINEs) are highly abundant in mammalian genomes. The term SINE has come to be restricted to short retroposons with internal RNA polymerase III promoter sites in a region derived from a structural RNA (usually a tRNA). Here we describe a novel, 260 bp tRNA-derived SINE, some fragments of which have been noted before to be repetitive in mammalian DNA. Unlike previously reported SINEs, which are restricted to closely related species, copies of this element can be found in all mammalian genomes, including marsupials. It is therefore called MIR for mammalian-wide interspersed repeat. Their high divergence and their presence at orthologous sites in different mammals indicate that MIRs, at least in part, amplified before the mammalian radiation. Next to Alu, MIRs are the most common interspersed repeat in primates with an estimated 300,000 copies still discernible, which account for 1 to 2% of our DNA. Interestingly, a small, central region of MIR appears to be much better conserved in the genomic copies than the rest of the sequence.

Animals↗

PubMed2003

Synthesis of adenosine derivatives as transcription initiators and preparation of 5' fluorescein- and biotin-labeled RNA through one-step in vitro transcription.

Expanding our previous finding of an adenosine-initiated transcription system, we now demonstrate that either the 5' site or the N6 site of adenosine nucleotides can be modified extensively without abolishing their ability to initiate transcription under the T7 phi2.5 promoter. Two series of amino derivatives of adenosine nucleotides were synthesized. Fluorescein and biotin groups were coupled to AMP derivatives through linkers of different sizes and hydrophobicities. Both fluorescein- and biotin-conjugated (at either the 5' or N6 site) adenosine nucleotides can act as efficient transcription initiators, producing fluorescein- and biotin-labeled RNA at the specific 5' end by a one-step transcription procedure, eliminating posttranscriptional modification. Furthermore, N6-modified adenosine derivative-initiated transcription synthesizes 5' end modified RNA with a free phosphate group, providing the possibility for further derivatization. The current finding makes easily available a variety of site-specifically functionalized RNA, which may be used in nucleic acid detection, RNA structural and functional investigation, and generation and isolation of novel functional RNA.

Adenosine↗

PubMed1997

[Structure and function of influenza virus nucleoprotein (NP)].

Influenza virus nucleoprotein (NP) is the major structural protein which interacts with the 8 segment RNAs, to make the panhandle structure of RNPs, nucleocapsids, together with 3 polymerase protein complex (PB2, PB1, and PA). The NP proteins (498 aminoacids, MW 56,000) self-polymerize to form the panhandle structure similarly as the nucleocapsids. The nucleocapsids ensure the base pairing RNA structure in the handle region corresponding to the complementary structure in the both ends of vRNAs. This handle structure is supposed to play roles in regulation of switch for transcription and replication, and encapsidation. The NP protein is one of the type-specific antigens distinguishing among influenza A, B, and C type, and a target of cross-reactive CTL.

Nucleocapsid↗

PubMed2001

Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3' splice site and its cis-regulatory element: possible involvement in RNA splicing.

The HIV-1 transcript is alternatively spliced to over 30 different mRNAs. Whether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary structure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A4b, A4c and A5 3' splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem-loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing silencer (ESS2) acting on site A3 is located in a long irregular stem-loop structure (SLS3). Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putative SR protein binding site. The other one is adjacent to ESS2. Unexpectedly, only the 3' A residue of ESS2 was protected. The suboptimal A3 polypyrimidine tract (PPT) is base paired. Using site-directed mutagenesis and transfection of a mini-HIV-1 cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an optimized PPT. Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.

3' Untranslated Regions↗

PubMed1998

The 5' and 3' TAR elements of human immunodeficiency virus exert effects at several points in the virus life cycle.

The human immunodeficiency virus type 1 RNA genome contains a terminal repeat (R) sequence that encodes the TAR hairpin motif, which has been implicated in Tat-mediated activation of transcription. More recently, a variety of other functions have been proposed for this structured RNA element. To determine the replicative roles of the 5' and 3' TAR hairpins, we analyzed multiple steps in the life cycle of wild-type and mutant viruses. A structure-destabilizing mutation was introduced in either the 5', the 3', or both TAR motifs of the proviral genome. As expected, opening of the 5' TAR hairpin caused a transcription defect. Because the level of protein expression was not similarly reduced, the translation of this mRNA was improved. No effect of the 3' hairpin on transcription and translation was measured. Mutations of the 5' and 3' hairpin structures reduced the efficiency of RNA packaging to similar extents, and RNA packaging was further reduced in the 5' and 3' TAR double mutant. Upon infection of cells with these virions, a reduced amount of reverse transcription products was synthesized by the TAR mutant. However, no net reverse transcription defect was observed after correction for the reduced level of virion RNA. This result was confirmed in in vitro reverse transcription assays. These data indicate that the 5' and 3' TAR motifs play important roles in several steps of the replication cycle, but these structures have no significant effect on the mechanism of reverse transcription.

Base Sequence↗

PubMed1976

Correlation of structural transitions in coliphage R17 with its loss of infectivity.

A circular dichroism comparative study of isolated and in situ phage R17 RNA reveals in both cases the same degree of base pairing. However, thermal circular dichroism melting profiles exhibit the presence of free energy of interaction between RNA and capsid protein. It is apparent that the capsid stabilizes the RNA structure with and without the addition of Mg2+. A close RNA capsid association is also derived from pH titration circular dichroism studies. The pH melting of the RNA in situ starts to occur about 0-5 pH unit higher with and without the addition of Mg2+ than the acid denaturation of isolated RNA. A direct correlation between bathochromic CD peak shift to the main position band and loss of survivors is noted for the thermal melting as well as pH titration experiments. It is suggested that the heat and pH induced conformational alterations of R17 RNA in situ coinciding with loss of infectivity occur after an in situ alteration of nucleic acid-capsid protein interaction.

Capsid↗

PubMed2006

Short, synthetic and selectively 13C-labeled RNA sequences for the NMR structure determination of protein-RNA complexes.

We report an optimized synthesis of all canonical 2'-O-TOM protected ribonucleoside phosphoramidites and solid supports containing [13C5]-labeled ribose moieties, their sequence-specific introduction into very short RNA sequences and their use for the structure determination of two protein-RNA complexes. These specifically labeled sequences facilitate RNA resonance assignments and are essential to assign a high number of sugar-sugar and intermolecular NOEs, which ultimately improve the precision and accuracy of the resulting structures. This labeling strategy is particularly useful for the study of protein-RNA complexes with single-stranded RNA in solution, which is rapidly an increasingly relevant research area in biology.

Carbon Isotopes↗

PubMed2000

A stem-loop motif formed by the immediate 5' terminus of the bovine viral diarrhea virus genome modulates translation as well as replication of the viral RNA.

Bovine viral diarrhea virus (BVDV), a Pestivirus member of the Flaviviridae family, has a positive-stranded RNA genome which consists of a single open reading frame (ORF) and untranslated regions (UTRs) at the 5' and 3' ends. The 5' UTR harbors extensive RNA structure motifs; most of them were shown to contribute to an internal ribosomal entry site (IRES), which mediates cap-independent translation of the ORF. The extreme 5'-terminal region of the BVDV genome had so far been believed not to be required for IRES function. By structure probing techniques, we initially verified the existence of a computer-predicted stem-loop motif at the 5' end of the viral genome (hairpin Ia) as well as at the 3' end of the complementary negative-strand replication intermediate [termed hairpin Ia (-)]. While the stem of this structure is mainly constituted of nucleotides that are conserved among pestiviruses, the loop region is predominantly composed of variable residues. Taking a reverse genetics approach to a subgenomic BVDV replicon RNA (DI9c) which could be equally employed in a translation as well as replication assay system based on BHK-21 cells, we obtained the following results. (i) Proper folding of the Ia stem was found to be crucial for efficient translation. Thus, in the context of an authentic replication-competent viral RNA, the 5'-terminal motif operates apparently as an integral functional part of the ribosome entry. (ii) An intact loop structure and a stretch of nucleotide residues that constitute a portion of the stem of the Ia or the Ia (-) motif, respectively, were defined to represent important determinants of the RNA replication pathway. (iii) Formation of the stem structure of the Ia (-) motif was determined to be not critical for RNA replication. In summary, our findings affirmed that the 5'-terminal region of the BVDV genome encodes a bifunctional secondary structure motif which may enable the viral RNA to switch from the translation to the replicative cycle and vice versa.

5' Untranslated Regions↗

PubMed2003

Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure.

DsrA RNA regulates the translation of two global regulatory proteins in Escherichia coli. DsrA activates the translation of RpoS while repressing the translation of H-NS. The RNA-binding protein Hfq is necessary for DsrA to function in vivo. Although Hfq binds to DsrA in vitro, the role of Hfq in DsrA-mediated regulation is not known. One hypothesis was that Hfq acts as an RNA chaperone by unfolding DsrA, thereby facilitating interactions with target RNAs. To test this hypothesis, we have examined the structure of DsrA bound to Hfq in vitro. Comparison of free DsrA to DsrA bound to Hfq by RNase footprinting, circular dichroism, and thermal melt profiles shows that Hfq does not alter DsrA secondary structures, but might affect its tertiary conformation. We identify the site on DsrA where Hfq binds, which is a structural element in the middle of DsrA. In addition, we show that although long poly(U) RNAs compete with DsrA for binding to Hfq, a short poly(U) stretch present in DsrA is not necessary for Hfq binding. Finally, unlike other RNAs, DsrA binding to Hfq is not competed with by poly(A) RNA. In fact, DsrA:poly(A):Hfq may form a stable ternary complex, raising the possibility that Hfq has multiple RNA-binding sites.

Bacterial Outer Membrane Proteins↗

PubMed1992

Cardioviral internal ribosomal entry site is functional in a genetically engineered dicistronic poliovirus.

High mutation rates have driven RNA viruses to shorten their genomes to the minimum possible size. Mammalian (+)-strand RNA viruses and retroviruses have responded by reducing the number of cis-acting regulatory elements, a constraint that has led to the emergence of the polyprotein. Poliovirus is a (+)-stranded picornavirus whose polyprotein, encoded by an open reading frame spanning most of the viral RNA, is processed by virus-encoded proteinases. Despite their genetic austerity, picornaviruses have retained long 5' untranslated regions, which harbour cis-acting elements that promote initiation of translation independently of the uncapped 5' end of the viral messenger RNA. These elements are termed 'internal ribosomal entry sites' and are formed from highly structured RNA segments of at least 400 nucleotides. How these elements function is not known, but special RNA-binding proteins may be involved. The ribosome or its 40S subunit probably binds at or near a YnXmAUG motif (where Y is a pyrimidine and X is a purine) at the 3' border of the internal ribosomal entry site, which either provides the initiating codon or enables the ribosome to translocate to one downstream (E.W. et al., submitted). Initiation from most eukaryotic messenger RNAs usually occurs by ribosomal recognition of the 5' and subsequent scanning to the AUG codon. Here we describe a genetic strategy for the dissection of polyproteins which proves that an internal ribosomal entry site element can initiate translation independently of the 5' end.

Binding Sites↗

PubMed2002

Structural basis of perturbed pKa values of catalytic groups in enzyme active sites.

In protein and RNA macromolecules, only a limited number of different side-chain chemical groups are available to function as catalysts. The myriad of enzyme-catalyzed reactions results from the ability of most of these groups to function either as nucleophilic, electrophilic, or general acid-base catalysts, and the key to their adapted chemical function lies in their states of protonation. Ionization is determined by the intrinsic pKa of the group and the microenvironment created around the group by the protein or RNA structure, which perturbs its intrinsic pKa to its functional or apparent pKa. These pKa shifts result from interactions of the catalytic group with other fully or partially charged groups as well as the polarity or dielectric of the medium that surrounds it. The electrostatic interactions between ionizable groups found on the surface of macromolecules are weak and cause only slight pKa perturbations (<2 units). The sum of many of these weak electrostatic interactions helps contribute to the stability of native or folded macromolecules and their ligand complexes. However, the pKa values of catalytic groups that are found in the active sites of numerous enzymes are significantly more perturbed (>2 units) and are the subject of this review. The magnitudes of these pKa perturbations are analyzed with respect to the structural details of the active-site microenvironment and the energetics of the reactions that they catalyze.

Binding Sites↗

PubMed1998

Structure of T7 RNA polymerase complexed to the transcriptional inhibitor T7 lysozyme.

The T7 RNA polymerase-T7 lysozyme complex regulates phage gene expression during infection of Escherichia coli. The 2.8 A crystal structure of the complex reveals that lysozyme binds at a site remote from the polymerase active site, suggesting an indirect mechanism of inhibition. Comparison of the T7 RNA polymerase structure with that of the homologous pol I family of DNA polymerases reveals identities in the catalytic site but also differences specific to RNA polymerase function. The structure of T7 RNA polymerase presented here differs significantly from a previously published structure. Sequence similarities between phage RNA polymerases and those from mitochondria and chloroplasts, when interpreted in the context of our revised model of T7 RNA polymerase, suggest a conserved fold.

Amino Acid Sequence↗

PubMed1991

Translational potentiation of messenger RNA with secondary structure in Xenopus.

Differential translation of messenger RNA (mRNA) with stable secondary structure in the 5' untranslated leader may contribute to the dramatic changes in protein synthetic patterns that occur during oogenesis and early development. Plasmids that contained the bacterial gene chloramphenicol acetyltransferase and which encoded mRNA with (hpCAT) or without (CAT) a stable hairpin secondary structure in the 5' noncoding region were transcribed in vitro, and the resulting mRNAs were injected into Xenopus oocytes, eggs, and early embryos. During early oogenesis, hpCAT mRNA was translated at less than 3 percent of the efficiency of CAT mRNA. The relative translational potential of hpCAT reached 100 percent in the newly fertilized egg and returned to approximately 3 percent after the midblastula transition.

Animals↗

PubMedDate not supplied

Small-angle X-ray and light scattering studies on the influence of Mg2+ ions on the structure of the RNA from bacteriophage MS2.

The influence of Mg2+ ions on the secondary and tertiary structure of the RNA from bacteriophage MS2 was investigated by small-angle X-ray scattering and light scattering and by sedimentation experiments. The analysis of the outer part of the X-ray scattering curve obtained at low temperature in the absence of Mg2+ yielded a cross-section radius of gyration of 0.88 nm and a mass per unit length of 1720 g mol-1 nm-1. Very similar values for these parameters, which refer to the secondary structure of the RNA molecule, were also derived from the X-ray scattering curves obtained in the presence of different amounts of Mg2+ (0.07 to 1 ions per nucleotide). On the contrary, the inner part of the X-ray scattering curves turned out to be highly dependent on the Mg2+ concentration: the cross-section radius of gyration and the mass per unit length, which were determined from the scattering curves at small angles as parameters related to the tertiary structure of the RNA, amounted to 3.11 nm and 4000 g mol-1 nm-1, respectively, in the absence of Mg2+ and increased significantly upon raising the concentration of Mg2+. The increase of these structural parameters was found to be accompanied by a decrease of the overall radius of gyration (as revealed indirectly by X-ray scattering and directly by light scattering measurements) and by an increase of the sedimentation coefficient. The results from the investigations of the RNA at low temperature clearly establish the existence of double-stranded structures down to very low Mg2+ concentrations as well as the occurrence of Mg2+ induced changes of the tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophages↗