scholarly journals Reverse Transcription of a Self-Primed Retrotransposon Requires an RNA Structure Similar to the U5-IR Stem-Loop of Retroviruses

1998 ◽  
Vol 18 (11) ◽  
pp. 6859-6869 ◽  
Author(s):  
Jia-Hwei Lin ◽  
Henry L. Levin
Keyword(s):  
Reverse Transcription ◽  
Rna Structure ◽  
Unique Feature ◽  
Gc Content ◽  
Specific Sequence ◽  
Stem Loop ◽  
Rous Sarcoma ◽  
Large Loop ◽  
Sarcoma Virus ◽  
And Function

ABSTRACT An inverted repeat (IR) within the U5 region of the Rous sarcoma virus (RSV) mRNA forms a structure composed of a 7-bp stem and a 5-nucleotide (nt) loop. This U5-IR structure has been shown to be required for the initiation of reverse transcription. The mRNA of Tf1, long terminal repeat-containing retrotransposon from fission yeast (Schizosaccharomyces pombe) contains nucleotides with the potential to form a U5-IR stem-loop that is strikingly similar to that of RSV. The putative U5-IR stem-loop of Tf1 consists of a 7-bp stem and a 25-nt loop. Results from mutagenesis studies indicate that the U5-IR stem-loop in the mRNA of Tf1 does form and that it is required for Tf1 transposition. Although the loop is required for transposition, we were surprised that the specific sequence of the nucleotides within the loop was unimportant for function. Additional investigation indicates that the loss of transposition activity due to a reduction in the loop size to 6 nt could be rescued by increasing the GC content of the stem. This result indicates that the large loop in the Tf1 mRNA relative to that of the RSV allows the formation of the relatively weak U5-IR stem. The levels of Tf1 proteins expressed and the amounts of Tf1 RNA packaged into the virus-like particles were not affected by mutations in the U5-IR structure. However, all of the mutations in the U5-IR structure that caused defects in transposition produced low amounts of reverse transcripts. A unique feature in the initiation of Tf1 reverse transcription is that, instead of a tRNA, the first 11 nt of the Tf1 mRNA serve as the minus-strand primer. Analysis of the 5′ end of Tf1 mRNA revealed that the mutations in the U5-IR stem-loop that resulted in defects in reverse transcription caused a reduction in the cleavage activity required to generate the Tf1 primer. Our results indicate that the U5-IR stems of Tf1 and RSV are conserved in size, position, and function.

scholarly journals Stem–loop formation drives RNA folding in mechanical unzipping experiments

2022 ◽  
Vol 119 (3) ◽  
pp. e2025575119
Author(s):  
Paolo Rissone ◽  
Cristiano V. Bizarro ◽  
Felix Ritort
Keyword(s):  
Optical Tweezers ◽  
Rna Structure ◽  
Nearest Neighbor ◽  
Rna Folding ◽  
General Mechanism ◽  
Loop Formation ◽  
Stem Loop ◽  
Wide Range ◽  
Irreversible Effects ◽  
And Function

Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive the 10 nearest-neighbor base pair (NNBP) RNA free energies in sodium and magnesium with 0.1 kcal/mol precision using optical tweezers. Notably, force–distance curves (FDCs) exhibit strong irreversible effects with hysteresis and several intermediates, precluding the extraction of the NNBP energies with currently available methods. The combination of a suitable RNA synthesis with a tailored pulling protocol allowed us to obtain the fully reversible FDCs necessary to derive the NNBP energies. We demonstrate the equivalence of sodium and magnesium free-energy salt corrections at the level of individual NNBP. To characterize the irreversibility of the unzipping–rezipping process, we introduce a barrier energy landscape of the stem–loop structures forming along the complementary strands, which compete against the formation of the native hairpin. This landscape correlates with the hysteresis observed along the FDCs. RNA sequence analysis shows that base stacking and base pairing stabilize the stem–loops that kinetically trap the long-lived intermediates observed in the FDC. Stem–loops formation appears as a general mechanism to explain a wide range of behaviors observed in RNA folding.

scholarly journals Structural Characterization of the Rous Sarcoma Virus RNA Stability Element

2008 ◽  
Vol 83 (5) ◽  
pp. 2119-2129 ◽  
Author(s):  
Jason E. Weil ◽  
Michalis Hadjithomas ◽  
Karen L. Beemon
Keyword(s):  
Secondary Structure ◽  
Rous Sarcoma Virus ◽  
Translation Termination ◽  
Rna Stability ◽  
Premature Termination Codon ◽  
Termination Codon ◽  
Stem Loop ◽  
Future Design ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT In eukaryotic cells, an mRNA bearing a premature termination codon (PTC) or an abnormally long 3′ untranslated region (UTR) is often degraded by the nonsense-mediated mRNA decay (NMD) pathway. Despite the presence of a 5- to 7-kb 3′ UTR, unspliced retroviral RNA escapes this degradation. We previously identified the Rous sarcoma virus (RSV) stability element (RSE), an RNA element downstream of the gag natural translation termination codon that prevents degradation of the unspliced viral RNA. Insertion of this element downstream of a PTC in the RSV gag gene also inhibits NMD. Using partial RNase digestion and selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, we determined the secondary structure of this element. Incorporating RNase and SHAPE data into structural prediction programs definitively shows that the RSE contains an AU-rich stretch of about 30 single-stranded nucleotides near the 5′ end and two substantial stem-loop structures. The overall secondary structure of the RSE appears to be conserved among 20 different avian retroviruses. The structural aspects of this element will serve as a tool in the future design of cis mutants in addressing the mechanism of stabilization.

Analysis of Adhesion Plaque Structure and Function in the Mechanism of Transformation by Rous Sarcoma Virus

1982 ◽  
Vol 40 ◽  
pp. 110-113
Author(s):  
L.R. Rohrschneider ◽  
M.J. Rosok ◽  
L.E. Gentry
Keyword(s):  
Rous Sarcoma Virus ◽  
Mammalian Cells ◽  
Neoplastic Transformation ◽  
Structure And Function ◽  
Excellent Opportunity ◽  
Cells In Culture ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
And Function ◽  
Adhesion Plaque

Rous sarcoma virus (RSV) was originally isolated from a fibrosarcoma of a chicken. This virus also will efficiently infect and transform all avian cells in culture as well as most mammalian cells. The mechanism of transformation by RSV is therefore universal and this system offers an excellent opportunity to investigate the mechanism of neoplastic transformation.

scholarly journals Cryo-EM structure of the Rous sarcoma virus octameric cleaved synaptic complex intasome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Krishan K. Pandey ◽  
Sibes Bera ◽  
Ke Shi ◽  
Michael J. Rau ◽  
Amarachi V. Oleru ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Single Amino Acid ◽  
Strand Transfer ◽  
Synaptic Complex ◽  
Rous Sarcoma ◽  
Tetrameric Structure ◽  
Sarcoma Virus ◽  
And Function ◽  
Key Steps

AbstractDespite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes.

Analysis of rous sarcoma virus (RSV) RNA structure by means of specific nucleases

Virology ◽  
1978 ◽  
Vol 90 (2) ◽  
pp. 317-329 ◽  
Author(s):  
Jean-L. Darlix ◽  
Pierre-F. Spahr ◽  
Peter A. Bromley
Keyword(s):  
Rous Sarcoma Virus ◽  
Rna Structure ◽  
Rous Sarcoma ◽  
Sarcoma Virus

scholarly journals Changes in Rous Sarcoma Virus RNA Secondary Structure near the Primer Binding Site upon tRNATrpPrimer Annealing

1999 ◽  
Vol 73 (8) ◽  
pp. 6307-6318 ◽  
Author(s):  
Shannon Morris ◽  
Jonathan Leis
Keyword(s):  
Binding Site ◽  
Rous Sarcoma Virus ◽  
Primer Binding Site ◽  
Wild Type ◽  
Stem Loop ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Primer Annealing ◽  
Nuclease Mapping

ABSTRACT Predicted secondary-structure elements encompassing the primer binding site in the 5′ untranslated region of Rous sarcoma virus (RSV) RNA play an integral role in multiple viral replications steps including reverse transcription, DNA integration, and RNA packaging (A. Aiyar, D. Cobrinik, Z. Ge, H. J. Kung, and J. Leis, J. Virol. 66:2464–2472, 1992; D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864–3872, 1991; J. T. Miller, Z. Ge, S. Morris, K. Das, and J. Leis, J. Virol. 71:7648–7656, 1997). These elements include the U5-Leader stem, U5-IR stem-loop, and U5-TΨC interaction region. Limited digestion of the 5′ untranslated region of wild-type and mutant RSV RNAs with structure- and/or sequence-specific RNases detects the presence of the U5-Leader stem and the U5-IR stem-loop. When a tRNATrp primer is annealed to wild-type RNAs in vitro, limited nuclease mapping indicates that the U5-IR stem becomes partially unwound. This is not observed when mutant RNAs with altered U5-IR stem-loop structures are substituted for wild-type RNAs. The U5-Leader stem also becomes destabilized when the tRNA primer is annealed to either wild-type or mutant RNA fragments. Nuclease mapping studies of tRNATrp, as well as the viral RNA, indicate that the U5-TΨC helix does form in vitro upon primer annealing. Collectively, these data suggest that the various structural elements near the RSV primer binding site undergo significant changes during the process of primer annealing.

scholarly journals The Effects of Alternate Polypurine Tracts (PPTs) and Mutations of Sequences Adjacent to the PPT on Viral Replication and Cleavage Specificity of the Rous Sarcoma Virus Reverse Transcriptase

2008 ◽  
Vol 82 (17) ◽  
pp. 8592-8604 ◽  
Author(s):  
Kevin W. Chang ◽  
Jangsuk Oh ◽  
W. Gregory Alvord ◽  
Stephen H. Hughes
Keyword(s):  
Reverse Transcriptase ◽  
Rous Sarcoma Virus ◽  
Point Mutations ◽  
Rnase H ◽  
Wild Type ◽  
Viral Dna ◽  
Specific Sequence ◽  
Cleavage Specificity ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT We previously reported that a mutant Rous sarcoma virus (RSV) with an alternate polypurine tract (PPT), DuckHepBFlipPPT, had unexpectedly high titers and that the PPT was miscleaved primarily at one position following a GA dinucleotide by the RNase H of reverse transcriptase (RT). This miscleavage resulted in a portion of the 3′ end of the PPT (5′-ATGTA) being added to the end of U3 of the linear viral DNA. To better understand the RNase H cleavage by RSV RT, we made a number of mutations within the DuckHepBFlipPPT and in the sequences adjacent to the PPT. Deleting the entire ATGTA sequence from the DuckHepBFlipPPT increased the relative titer to wild-type levels, while point mutations within the ATGTA sequence reduced the relative titer but had minimal effects on the cleavage specificity. However, mutating a sequence 5′ of ATGTA affected the relative titer of the virus and caused the RNase H of RSV RT to lose the ability to cleave the PPT specifically. In addition, although mutations in the conserved stretch of thymidine residues upstream of the PPT did not affect the relative titer or cleavage specificity, the mutation of some of the nucleotides immediately upstream of the PPT did affect the titer and cleavage specificity. Taken together, our studies show that the structure of the PPT in the context of the cognate RT, rather than a specific sequence, is important for the proper cleavage by RSV RT.

scholarly journals The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA

2019 ◽  
Vol 48 (2) ◽  
pp. 830-846 ◽  
Author(s):  
Rita Pinto ◽  
Cathrine B Vågbø ◽  
Magnus E Jakobsson ◽  
Yeji Kim ◽  
Marijke P Baltissen ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Rna Structure ◽  
Mrna Translation ◽  
28S Rrna ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Reversible Modification ◽  
And Shifts ◽  
And Function

Abstract RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem–loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.

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