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PubMed2006

Long-distance RNA-RNA interactions between terminal elements and the same subset of internal elements on the potato virus X genome mediate minus- and plus-strand RNA synthesis.

Potexvirus genomes contain conserved terminal elements that are complementary to multiple internal octanucleotide elements. Both local sequences and structures at the 5' terminus and long-distance interactions between this region and internal elements are important for accumulation of potato virus X (PVX) plus-strand RNA in vivo. In this study, the role of the conserved hexanucleotide motif within SL3 of the 3' NTR and internal conserved octanucleotide elements in minus-strand RNA synthesis was analyzed using both a template-dependent, PVX RNA-dependent RNA polymerase (RdRp) extract and a protoplast replication system. Template analyses in vitro indicated that 3' terminal templates of 850 nucleotides (nt), but not 200 nt, supported efficient, minus-strand RNA synthesis. Mutational analyses of the longer templates indicated that optimal transcription requires the hexanucleotide motif in SL3 within the 3' NTR and the complementary CP octanucleotide element 747 nt upstream. Additional experiments to disrupt interactions between one or more internal conserved elements and the 3' hexanucleotide element showed that long-distance interactions were necessary for minus-strand RNA synthesis both in vitro and in vivo. Additionally, multiple internal octanucleotide elements could serve as pairing partners with the hexanucleotide element in vivo. These cis-acting elements and interactions correlate in several ways to those previously observed for plus-strand RNA accumulation in vivo, suggesting that dynamic interactions between elements at both termini and the same subset of internal octanucleotide elements are required for both minus- and plus-strand RNA synthesis and potentially other aspects of PVX replication.

Base Sequence↗

PubMed2006

The importance of RNA structure in RNA editing and a potential proofreading mechanism for correct guide RNA:pre-mRNA binary complex formation.

RNA editing in trypanosomes is a post-transcriptional process responsible for correcting the coding sequences of many mitochondrial mRNAs. Uridine bases are specifically added or deleted from mRNA by an enzymatic cascade in which a pre-edited mRNA is cleaved specifically, uridine bases are added or removed, and the corrected mRNA is ligated. The process is directed by RNA molecules, termed guide RNAs (gRNA). The ability of this class of small, non-coding RNA to function in RNA editing is essential for these organisms. Typically, gRNAs are transcribed independent of their cognate mRNA and anneal to form a binary RNA complex. An exception from this process is the cytochrome oxidase subunit II (COII) mRNA, which encodes its gRNA within its 3' untranslated region. This gRNA lacks the ability to function in trans. Using an in vitro editing assay, we find that improving thermodynamic stability to the anchor region through increased Watson-Crick base-pairing is sufficient to impart trans editing activity. We further show that a point mutation outside the known functional regions of a gRNA induces both a conformational rearrangement of the gRNA and causes a decrease in the rate of editing. Taken together, these results lead us to propose a model for a potential proofreading step in the formation of a gRNA:pre-edited mRNA binary complex. The mechanism relies on the thermodynamic stability supplied to the RNA complex through Watson-Crick base-pairing. Through mutations in the gRNA, we demonstrate the importance of gRNA structure to the RNA editing reaction.

3' Untranslated Regions↗

PubMed1995

Formation of brome mosaic virus RNA-dependent RNA polymerase in yeast requires coexpression of viral proteins and viral RNA.

In this report we show that yeast expressing brome mosaic virus (BMV) replication proteins 1a and 2a and replicating a BMV RNA3 derivative can be extracted to yield a template-dependent BMV RNA-dependent RNA polymerase (RdRp) able to synthesize (-)-strand RNA from BMV (+)-strand RNA templates added in vitro. This virus-specific yeast-derived RdRp mirrored the template selectivity and other characteristics of RdRp from BMV-infected plants. Equivalent extracts from yeast expressing 1a and 2a but lacking RNA3 contained normal amounts of 1a and 2a but had no RdRp activity on BMV RNAs added in vitro. To determine which RNA3 sequences were required in vivo to yield RdRp activity, we tested deletions throughout RNA3, including the 5',3', and intercistronic noncoding regions, which contain the cis-acting elements required for RNA3 replication in vivo. RdRp activity was obtained only from cells expressing 1a, 2a, and RNA3 derivatives retaining both 3' and intercistronic noncoding sequences. Strong correlation between extracted RdRp activity and BMV (-)-strand RNA accumulation in vivo was found for all RNA3 derivatives tested. Thus, extractable in vitro RdRp activity paralleled formation of a complex capable of viral RNA synthesis in vivo. The results suggest that assembly of active RdRp requires not only viral proteins but also viral RNA, either to directly contribute some nontemplate function or to recruit essential host factors into the RdRp complex and that sequences at both the 3'-terminal initiation site and distant internal sites of RNA3 templates may participate in RdRp assembly and initiation of (-)-strand synthesis.

Bromovirus↗

PubMed2001

In vitro RNA synthesis from exogenous dengue viral RNA templates requires long range interactions between 5'- and 3'-terminal regions that influence RNA structure.

Viral replicases of many positive-strand RNA viruses are membrane-bound complexes of cellular and viral proteins that include viral RNA-dependent RNA polymerase (RdRP). The in vitro RdRP assay system that utilizes cytoplasmic extracts from dengue viral-infected cells and exogenous RNA templates was developed to understand the mechanism of viral replication in vivo. Our results indicated that in vitro RNA synthesis at the 3'-untranslated region (UTR) required the presence of the 5'-terminal region (TR) and the two cyclization (CYC) motifs suggesting a functional interaction between the TRs. In this study, using a psoralen-UV cross-linking method and an in vitro RdRP assay, we analyzed structural determinants for physical and functional interactions. Exogenous RNA templates that were used in the assays contained deletion mutations in the 5'-TR and substitution mutations in the 3'-stem-loop structure including those that would disrupt the predicted pseudoknot structure. Our results indicate that there is physical interaction between the 5'-TR and 3'-UTR that requires only the CYC motifs. RNA synthesis at the 3'-UTR, however, requires long range interactions involving the 5'-UTR, CYC motifs, and the 3'-stem-loop region that includes the tertiary pseudoknot structure.

3' Untranslated Regions↗

PubMed1990

B2 RNA and 7SK RNA, RNA polymerase III transcripts, have a cap-like structure at their 5' end.

We found that hydrolysates of poly(A)+ RNA from Ehrlich ascites carcinoma cells which were transcribed by RNA polymerase III contained an unusual component designated as X. It was part of B2 RNA representing a transcript of B2 retroposon, typical of rodents. The component X possesses a cap-like structure, xppp5'G, where x has a non-nucleotide structure. About half of all B2 RNAs contained this group at the 5' end. Previously, Epstein et al. (1) detected a similar structure at the 5' end of small nuclear U6 RNA. Later, Singh and Reddy (2) showed methyl to be the blocking group in the component x of U6 RNA. Besides B2 RNA, we found 5' ends containing methyl groups in 7SK RNA.

Animals↗

PubMed1988

RNA as an RNA polymerase: net elongation of an RNA primer catalyzed by the Tetrahymena ribozyme.

A catalytic RNA (ribozyme) derived from an intervening sequence (IVS) RNA of Tetrahymena thermophila will catalyze an RNA polymerization reaction in which pentacytidylic acid (C5) is extended by the successive addition of mononucleotides derived from a guanylyl-(3',5')-nucleotide (GpN). Cytidines or uridines are added to C5 to generate chain lengths of 10 to 11 nucleotides, with longer products being generated at greatly reduced efficiency. The reaction is analogous to that catalyzed by a replicase with C5 acting as the primer, GpNs as the nucleoside triphosphates, and a sequence in the ribozyme providing a template. The demonstration that an RNA enzyme can catalyze net elongation of an RNA primer supports theories of prebiotic RNA self-replication.

Animals↗

PubMed2005

The AAUAAA motif of bamboo mosaic virus RNA is involved in minus-strand RNA synthesis and plus-strand RNA polyadenylation.

Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome with a 5'-cap structure and a 3' poly(A) tail. Deleting the internal loop that contains the putative polyadenylation signal (AAUAAA) in the 3' untranslated region (UTR) of BaMV genomic RNA appeared to diminish coat protein accumulation to 2% (C. P. Cheng and C. H. Tsai, J. Mol. Biol. 288:555-565, 1999). To investigate the function of the AAUAAA motif, mutations were introduced into an infectious BaMV cDNA at each residue except the first nucleotide. After transfection of Nicotiana benthamiana protoplasts with RNA transcript, the accumulations of viral coat protein and RNAs were determined. Based on the results, three different categories could be deduced for the mutants. Category 1 includes two mutants expressing levels of the viral products similar to those of the wild-type virus. Six mutations in category 2 led to decreased to similar levels of both minus-strand and genomic RNAs. Category 3 includes the remaining seven mutations that also bring about decreases in both minus- and plus-strand RNA levels, with more significant effects on genomic RNA accumulation. Mutant transcripts from each category were used to infect N. benthamiana plants, from which viral particles were isolated. The genomic RNAs of mutants in category 3 were found to have shorter poly(A) tails. Taken together, the results suggest that the AAUAAA motif in the 3' UTR of BaMV genomic RNA is involved not only in the formation of the poly(A) tail of the plus-strand RNA, but also in minus-strand RNA synthesis.

3' Untranslated Regions↗

PubMed1991

Molecular cloning and characterization of W double-stranded RNA, a linear molecule present in Saccharomyces cerevisiae. Identification of its single-stranded RNA form as 20 S RNA.

Most strains of the yeast Saccharomyces cerevisiae harbor a double-stranded RNA (dsRNA) molecule, called W. We obtained W cDNA clones by random priming of denatured W dsRNA followed by reverse transcription. Sequence data of W shows that only one strand ((+)-strand) has coding capacity for a protein with 829 amino acids which spans almost the entire length of the molecule (2.5 kilobases). Within this protein we found a sequence pattern characteristic of RNA dependent RNA polymerases of (+)-strand and double-stranded RNA viruses. W has no homology with other dsRNAs found in S. cerevisiae, such as L-A, L-BC or M1. However, a (+)-strand-specific probe for W hybridized with 20-S RNA. Furthermore, W (+)-strands comigrated with 20 S RNA in strand separation gels. These results suggest that 20 S RNA is a (+)-single-stranded RNA form of W dsRNA itself or a closely related molecule.

Amino Acid Sequence↗

PubMed1990

Mutations affecting primer RNA interaction with the replication repressor RNA I in plasmid CoIE1: potential RNA folding pathway mutants.

The control of plasmid ColE1 copy number is mediated by the kinetics of interaction of two complementary plasmid-encoded RNAs. One RNA is the primer precursor and the other is a small counter-transcript called RNA I. The interaction of these highly structured RNAs results in inhibition of formation of mature primer RNA necessary for replication initiation. We have studied several plasmid copy number mutants which have single base changes in the primer which render the primer resistant to inhibition by RNA I despite the fact that the mutations are located outside the overlap between primer and RNA I. We propose a model to account for the resistance of the mutant primers which is based on the differential folding of the nascent primer transcripts during transcription. We propose that the mutant primers diverge in structure from their wild-type counterparts during a discrete period during transcription. During this brief divergence, they are proposed to interact kinetically more slowly with RNA I than wild-type primer because a particular domain (the anti-tail) required for efficient interaction with RNA I is buried in a stem-loop structure while this same domain is predicted to be single-stranded in the wild-type. Despite substantial sequence divergence from ColE1, the primer precursors of the related plasmids CloDF13, RSF1030 and p15A also have retained the potential to expose their anti-tail in a similar manner to ColE1, suggesting that the folding pathway has been conserved in evolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1997

The U5 RNA of trypanosomes deviates from the canonical U5 RNA: the Leptomonas collosoma U5 RNA and its coding gene.

Fractionation of the abundant small ribonucleoproteins (RNPs) of the trypanosomatid Leptomonas collosoma revealed the existence of a group of unidentified small RNPs that were shown to fractionate differently than the well-characterized trans-spliceosomal RNPs. One of these RNAs, an 80-nt RNA, did not possess a trimethylguanosine (TMG) cap structure but did possess a 5' phosphate terminus and an invariant consensus U5 snRNA loop 1. The gene coding for the RNA was cloned, and the coding region showed 55% sequence identity to the recently described U5 homologue of Trypanosoma brucei [Dungan, J. D., Watkins, K. P. & Agabian, N. (1996) EMBO J. 15, 4016-4029]. The L. collosoma U5 homologue exists in multiple forms of RNP complexes, a 10S monoparticle, and two subgroups of 18S particles that either contain or lack the U4 and U6 small nuclear RNAs, suggesting the existence of a U4/U6.U5 tri-small nuclear RNP complex. In contrast to T. brucei U5 RNA (62 nt), the L. collosoma homologue is longer (80 nt) and possesses a second stem-loop. Like the trypanosome U3, U6, and 7SL RNA genes, a tRNA gene coding for tRNACys was found 98 nt upstream to the U5 gene. A potential for base pair interaction between U5 and SL RNA in the 5' splice site region (positions -1 and +1) and downstream from it is proposed. The presence of a U5-like RNA in trypanosomes suggests that the most essential small nuclear RNPs are ubiquitous for both cis- and trans-splicing, yet even among the trypanosomatids the U5 RNA is highly divergent.

Amino Acid Sequence↗

PubMed1971

Turnover of HeLa ribosomal RNA. The characterization of a class of RNA in HeLa cytoplasm derived from 28S RNA.

A class of RNA in HeLa cytoplasm with a sedimentation value of 22S is described. This RNA is a true cytoplasmic component and is not an artifact of cell rupture or of the method of RNA preparation. The 22S RNA sediments in a sucrose gradient with those ribosomal structures containing 28S ribosomal RNA (60S and 74S particles and polyribosomes). It has a base composition and methyl content similar to those of 28S RNA. The kinetics of formation of 22S suggest that it is not a direct product of transcription but is derived in vivo from "old" molecules of 28S RNA.

Base Sequence↗

PubMed2000

Role of the 3' tRNA-like structure in tobacco mosaic virus minus-strand RNA synthesis by the viral RNA-dependent RNA polymerase In vitro.

A template-dependent RNA polymerase has been used to determine the sequence elements in the 3' untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3'-terminal 4 nucleotides of domain D1 indicated the importance of the 3'-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3' untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

3' Untranslated Regions↗

PubMed2002

Requirements for de novo initiation of RNA synthesis by recombinant flaviviral RNA-dependent RNA polymerases.

RNA-dependent RNA polymerases (RdRps) that initiate RNA synthesis by a de novo mechanism should specifically recognize the template initiation nucleotide, T1, and the substrate initiation nucleotide, the NTPi. The RdRps from hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), and GB virus-B all can initiate RNA synthesis by a de novo mechanism. We used RNAs and GTP analogs, respectively, to examine the use of the T1 nucleotide and the initiation nucleotide (NTPi) during de novo initiation of RNA synthesis. The effects of the metal ions Mg(2+) and Mn(2+) on initiation were also analyzed. All three viral RdRps require correct base pairing between the T1 and NTPi for efficient RNA synthesis. However, each RdRp had some distinct tolerances for modifications in the T1 and NTPi. For example, the HCV RdRp preferred an NTPi lacking one or more phosphates regardless of whether Mn(2+) was present or absent, while the BVDV RdRp efficiently used GDP and GMP for initiation of RNA synthesis only in the presence of Mn(2+). These and other results indicate that although the three RdRps share a common mechanism of de novo initiation, each has distinct preferences.

Bromovirus↗

PubMed2004

Membrane association of the RNA-dependent RNA polymerase is essential for hepatitis C virus RNA replication.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), belongs to a class of integral membrane proteins termed tail-anchored proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane protein-protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored proteins.

Amino Acid Sequence↗

PubMed2005

Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II.

Transcription by the influenza virus RNA-dependent RNA polymerase is dependent on cellular RNA processing activities that are known to be associated with cellular RNA polymerase II (Pol II) transcription, namely, capping and splicing. Therefore, it had been hypothesized that transcription by the viral RNA polymerase and Pol II might be functionally linked. Here, we demonstrate for the first time that the influenza virus RNA polymerase complex interacts with the large subunit of Pol II via its C-terminal domain. The viral polymerase binds hyperphosphorylated forms of Pol II, indicating that it targets actively transcribing Pol II. In addition, immunofluorescence analysis is consistent with a new model showing that influenza virus polymerase accumulates at Pol II transcription sites. The present findings provide a framework for further studies to elucidate the mechanistic principles of transcription by a viral RNA polymerase and have implications for the regulation of Pol II activities in infected cells.

Animals↗

PubMed2003

A 100-kD complex of two RNA-binding proteins from mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts with several RNA editing components.

A stable 100-kD complex from mitochondria of Leishmania tarentolae containing two RNA-binding proteins, Ltp26 and Ltp28, was identified by cross-linking to unpaired 4-thiouridine nucleotides in a partially duplex RNA substrate. The genes were cloned and expressed and the complex was reconstituted from recombinant proteins in the absence of RNA or additional factors. The Ltp26 and Ltp28 proteins are homologs of gBP27 and gBP29 from Crithidia fasciculata and gBP25 and gBP21 from Trypanosoma brucei, respectively. The purified Ltp26/Ltp28 complex, the individual recombinant proteins, and the reconstituted complex are each capable of catalyzing the annealing of complementary RNAs, as was previously shown for gBP21 from T. brucei. A high-molecular-weight RNP complex consisting of the Ltp26/Ltp28 complex and several 55-60-kD proteins together with guide RNA could be purified from mitochondrial extract of L. tarentolae transfected with Ltp28-TAP. This complex also interacted in a less stable manner with the RNA ligase-containing L-complex and with the 3' TUTase. The Ltp26/Ltp28 RNP complex is a candidate for catalyzing the annealing of guide RNA and pre-edited mRNA in the initial step of RNA editing.

Amino Acid Sequence↗

PubMed1995

Isolation and characterization of intranuclear ribonucleoprotein complexes associated with double-stranded RNA adenosine deaminase from brain cells: implications for RNA-editing and hypermutation of viral RNA in the CNS.

Double-stranded RNA adenosine deaminase (DsRAD), which converts adenosine in duplex RNA to inosine, has been implicated in editing of cellular mRNA and hypermutation of viral RNA in the central nervous system (CNS). We used subcellular fractionation to show that DsRAD in bovine brain tissues is associated with high-molecular-weight ribonucleoprotein (RNP) complexes in the nuclei. DsRAD-associated RNP complexes have apparent molecular mass of up to 500 kDa and buoyant density of 1.35 to 1.42 g cc-1 in CsCl solution. In human glioma cells, DsRAD is also found exclusively in intranuclear RNP complexes that co-sediment with the largest RNA species. These DsRAD-associated RNP complexes are dissociated by RNase A or high salt. The RNA component is not essential for DsRAD activity, and the protein component can be separated by dsRNA-affinity column, gel filtration column, and glycerol gradient into enzymatically active protein species with apparent molecular mass ranging from 120 kDa to 70 kDa in polyacrylamide gel. The bovine brain DsRAD has no apparent requirement for low-molecular-weight cofactors or metal ions. These results provide insight into the native state of DsRAD in brain cells and have interesting implications for its putative roles in RNA-editing and hypermutation of viral RNA in the CNS.

Adenosine Deaminase↗

PubMed2006

HIV-1 TAR RNA subverts RNA interference in transfected cells through sequestration of TAR RNA-binding protein, TRBP.

TAR RNA-binding protein, TRBP, was recently discovered to be an essential partner for Dicer and a crucial component of the RNA-induced silencing complex (RISC), a critical element of the RNA interference (RNAi) of the cell apparatus. Human TRBP was originally characterized and cloned 15 years ago based on its high affinity for binding the HIV-1 encoded leader RNA, TAR. RNAi is used, in part, by cells to defend against infection by viruses. Here, we report that transfected TAR RNA can attenuate the RNAi machinery in human cells. Our data suggest that TAR RNA sequesters TRBP rendering it unavailable for downstream Dicer-RISC complexes. TAR-induced inhibition of Dicer-RISC activity in transfected cells was partially relieved by exogenous expression of TRBP.

HIV Long Terminal Repeat↗