Chemical and Computer Probing of RNA Structure

Колчанов, Н. А.; Titov, I. I.; Vlassova, I. E.; Vlassov, V. V.

doi:10.1016/s0079-6603(08)60144-0

Cited by 27 publications

(10 citation statements)

References 152 publications

(2 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For the enzymatic structure probing, the single-stranded specific RNase T1 was used, which cleaves at the 3Ј of unpaired G residues, and RNase U2, which cleaves at the 3Ј of unpaired A or G with a preference for A's. For the chemical probing we used imidazole (27) and lead acetate (28), which are both single strandedspecific. Reactions were analyzed on denaturing gels of various acrylamide concentrations to maximize the information from each probing reaction.…”

Section: Rna Structure Mapping Of Fragments 1-206 and ␣3-mentioning

confidence: 99%

Sequence Specificity in the Interaction of Bluetongue Virus Non-structural Protein 2 (NS2) with Viral RNA

Lymperopoulos

Wirblich

Brierley

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

The non-structural protein NS2 of Bluetongue virus (BTV) is synthesized abundantly in virus-infected cells and has been suggested to be involved in virus replication. The protein, with a high content of charged residues, possesses a strong affinity for single-stranded RNA species but, to date, all studies have failed to identify any specificity in the NS2-RNA interaction. In this report, we have examined, through RNA binding assays using highly purified NS2, the specificity of interaction with different single-stranded RNA (ssRNA) species in the presence of appropriate competitors. The data obtained show that NS2 indeed has a preference for BTV ssRNA over nonspecific RNA species and that NS2 recognizes a specific region within the BTV10 segment S10. The secondary structure of this region was determined and found to be a hairpin-loop with substructures within the loop. Modification-inhibition experiments highlighted two regions within this structure that were protected from ribonuclease cleavage in the presence of NS2. Overall, these data imply that a function of NS2 may be to recruit virus messenger RNAs (that also act as templates for synthesis of genomic RNAs) selectively from other RNA species within the infected cytosol of the cell during virus replication.Viruses that have a segmented RNA genome face a challenging task in the recruitment and assortment of specific viralcoded RNA species in the infected cells. It is commonly believed that each of the viral segments must possess some specific sequence or structures, which is recognized by one or more virally encoded proteins to facilitate these processes. Generally, viral RNA-binding proteins are distinguished by being in one of two categories. The first includes proteins that are associated with the nucleocapsid (nucleoproteins) (1) and are involved in the replication process (transcription and packaging) of the viral genome (2, 3). The second includes proteins that play an essential role in recruiting, transporting (4), modifying (5), and translating (6) viral RNA. These proteins can also interact with cellular RNA to suppress the expression of regulatory genes (7), so protecting the viral RNA from cellular recognition mechanisms to use the cellular machinery for virus propagation (6). Members of the Reoviridae have segmented double-stranded RNA genomes enclosed within the double capsids of the virions. During virus entry into the host cells the outer capsid proteins are lost, allowing the viral core to initiate the transcription of genomic RNAs. The newly synthesized single-stranded RNA species are subsequently released into the cytosol and in turn serve both as templates for viral doublestranded RNA genome synthesis and also act as messengers for the synthesis of viral proteins within the cytoplasm (see review, see Ref. 8). However, to date it is not known how the 10 -12 RNA segments are specifically recruited and transported to the virus replication and assembly sites within the cytoplasm and if any specific sequence is involved. Bluetongue virus is an o...

show abstract

Section: Rna Structure Mapping Of Fragments 1-206 and ␣3-mentioning

confidence: 99%

Sequence Specificity in the Interaction of Bluetongue Virus Non-structural Protein 2 (NS2) with Viral RNA

Lymperopoulos

Wirblich

Brierley

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…RNase V1 is not base-specific but cleaves RNA that is in helical conformation, whether base-paired (a minimum of 4-6bp are required) or single stranded and stacked. We also used the single-strand-specific chemical probes imidazole (Vlassov et al, 1995) and lead acetate (Krzyzosiak et al, 1988;Kolchanov et al, 1996). In reactions with RG501, the compound was preincubated with the RNA for 10 minutes at 30°C prior to structure probing.…”

Section: Frameshifting As a Target For Antiviral Interventionmentioning

confidence: 99%

V, 2.Ribosomal frameshifting in astroviruses

Brierley

Vidakovic

2003

Perspectives in Medical Virology

View full text Add to dashboard Cite

“…RNase V 1 is not base-speci®c but cleaves RNA that is in helical conformation, whether base-paired (a minimum of 4-6 bp are required) or single-stranded and stacked. We also used the single strand-speci®c chemical probes imidazole (Vlassov et al, 1995) and lead acetate (Krzyzosiak et al, 1988;Kolchanov et al, 1996).…”

Section: Ibv-pseudoknot Induced à1 Frameshifting Requires a Minimum Smentioning

confidence: 99%

The role of RNA pseudoknot stem 1 length in the promotion of efficient −1 ribosomal frameshifting

Napthine

Liphardt

Bloys

et al. 1999

Journal of Molecular Biology

View full text Add to dashboard Cite

The ribosomal frameshifting signal present in the genomic RNA of the coronavirus infectious bronchitis virus (IBV) contains a classic hairpin-type RNA pseudoknot that is believed to possess coaxially stacked stems of 11 bp (stem 1) and 6 bp (stem 2). We investigated the influence of stem 1 length on the frameshift process by measuring the frameshift efficiency in vitro of a series of IBV-based pseudoknots whose stem 1 length was varied from 4 to 13 bp in single base-pair increments. Efficient frameshifting depended upon the presence of a minimum of 11 bp; pseudoknots with a shorter stem 1 were either non-functional or had reduced frameshift efficiency, despite the fact that a number of them had a stem 1 with a predicted stability equal to or greater than that of the wild-type IBV pseudoknot. An upper limit for stem 1 length was not determined, but pseudoknots containing a 12 or 13 bp stem 1 were fully functional. Structure probing analysis was carried out on RNAs containing either a ten or 11 bp stem 1; these experiments confirmed that both RNAs formed pseudoknots and appeared to be indistinguishable in conformation. Thus the difference in frameshifting efficiency seen with the two structures was not simply due to an inability of the 10 bp stem 1 construct to fold into a pseudoknot. In an attempt to identify other parameters which could account for the poor functionality of the shorter stem 1-containing pseudoknots, we investigated, in the context of the 10 bp stem 1 construct, the influence on frameshifting of altering the slippery sequence-pseudoknot spacing distance, loop 2 length, and the number of G residues at the bottom of the 5'-arm of stem 1. For each parameter, it was possible to find a condition where a modest stimulation of frameshifting was observable (about twofold, from seven to a maximal 17 %), but we were unable to find a situation where frameshifting approached the levels seen with 11 bp stem 1 constructs (48-57 %). Furthermore, in the next smaller construct (9 bp stem 1), changing the bottom four base-pairs to G.C (the optimal base composition) only stimulated frameshifting from 3 to 6 %, an efficiency about tenfold lower than seen with the 11 bp construct. Thus stem 1 length is a major factor in determining the functionality of this class of pseudoknot and this has implications for models of the frameshift process.

show abstract

Chemical and Computer Probing of RNA Structure

Abstract: Siberian Division of Russian Academy Novosibirsk 630090, Russia of Sciences +Institute of Bioorganic Chemistry of Sciences I. Probing RNA Structure by Chemical and Enzymatic Approaches . . . . .

Cited by 27 publications

References 152 publications

Sequence Specificity in the Interaction of Bluetongue Virus Non-structural Protein 2 (NS2) with Viral RNA

Sequence Specificity in the Interaction of Bluetongue Virus Non-structural Protein 2 (NS2) with Viral RNA

V, 2.Ribosomal frameshifting in astroviruses

The role of RNA pseudoknot stem 1 length in the promotion of efficient −1 ribosomal frameshifting

Contact Info

Product

Resources

About