Dorota Piekna-Przybylska

The 3D rRNA modification maps database: with interactive tools for ribosome analysis.

The 3D rRNA modification maps database is the first general resource of information about the locations of modified nucleotides within the 3D structure of the full ribosome, with mRNA and tRNAs in the A-, P- and E-sites. The database supports analyses for several model organisms, including higher eukaryotes, and enables users to construct 3D maps for other organisms. Data are provided for human and plant (Arabidopsis) ribosomes, and for other representative organisms from eubacteria, archaea and eukarya. Additionally, the database integrates information about positions of modifications within rRNA sequences and secondary structures, as well as links to other databases and resources about modifications and their biosynthesis. Displaying positions of modified nucleotides is fully manageable. Views of each modified nucleotide are controlled by individual buttons and buttons also control the visibility of different ribosomal molecular components. A section called ‘Paint Your Own’ enables the user to create a 3D modification map for rRNA from any organism where sites of modification are known. This section also provides capabilities for visualizing nucleotides of interest in rRNA or tRNA, as well as particular amino acids in ribosomal proteins. The database can be accessed at http://people.biochem.umass.edu/fournierlab/3dmodmap/

Ribosome performance is enhanced by a rich cluster of pseudouridines in the A-site finger region of the large subunit.

The large subunit rRNA in eukaryotes contains an unusually dense cluster of 8–10 pseudouridine (Ψ) modifications located in a three-helix structure (H37–H39) implicated in several functions. This region is dominated by a long flexible helix (H38) known as the “A-site finger” (ASF). The ASF protrudes from the large subunit just above the A-site of tRNA binding, interacts with 5 S rRNA and tRNA, and through the terminal loop, forms a bridge (B1a) with the small subunit. In yeast, the three-helix domain contains 10 Ψs and 6 are concentrated in the ASF helix (3 of the ASF Ψs are conserved among eukaryotes). Here, we show by genetic depletion analysis that the Ψs in the ASF helix and adjoining helices are not crucial for cell viability; however, their presence notably enhances ribosome fitness. Depleting different combinations of Ψs suggest that the modification pattern is important and revealed that loss of multiple Ψs negatively influences ribosome performance. The effects observed include slower cell growth (reduced rates up to 23% at 30 °C and 40–50% at 37 °C and 11 °C), reduced level of the large subunit (up to 17%), impaired polysome formation (appearance of half-mers), reduced translation activity (up to 20% at 30 °C and 25% at 11 °C), and increased sensitivity to ribosome-based drugs. The results indicate that the Ψs in the three-helix region improve fitness of a eukaryotic ribosome.

U3 Region in the HIV-1 Genome Adopts a G-Quadruplex Structure in Its RNA and DNA Sequence

Genomic regions rich in G residues are prone to adopt G-quadruplex structure. Multiple Sp1-binding motifs arranged in tandem have been suggested to form this structure in promoters of cancer-related genes. Here, we demonstrate that the G-rich proviral DNA sequence of the HIV-1 U3 region, which serves as a promoter of viral transcription, adopts a G-quadruplex structure. The sequence contains three binding elements for transcription factor Sp1, which is involved in the regulation of HIV-1 latency, reactivation, and high-level virus expression. We show that the three Sp1 binding motifs can adopt different forms of G-quadruplex structure and that the Sp1 protein can recognize and bind to its site folded into a G-quadruplex. In addition, a c-kit2 specific antibody, designated hf2, binds to two different G-quadruplexes formed in Sp1 sites. Since U3 is encoded at both viral genomic ends, the G-rich sequence is also present in the RNA genome. We demonstrate that the RNA sequence of U3 forms dimers with characteristics known for intermolecular G-quadruplexes. Together with previous reports showing G-quadruplex dimers in the gag and cPPT regions, these results suggest that integrity of the two viral genomes is maintained through numerous intermolecular G-quadruplexes formed in different RNA genome locations. Reconstituted reverse transcription shows that the potassium-dependent structure formed in U3 RNA facilitates RT template switching, suggesting that the G-quadruplex contributes to recombination in U3.

Mis-targeted methylation in rRNA can severely impair ribosome synthesis and activity

Eukaryotic rRNAs contain scores of two major types of nucleotide modifications, 2’-O-methylation (Nm) and pseudouridylation (Ψ). Both types are known to alter the stability and dynamics of RNA folding. In Eukaryotes, these modifications are created by small nucleolar RNPs (snoRNPs) with site-specificity provided by the snoRNA component. Little is yet known about the influence of such modifications on ribosome synthesis or activity, although in a few cases depletions of natural modifications have impaired ribosome function. Our previous work showed that targeting Nm modifications to non-natural sites in yeast rRNA can severely impair cell growth, however, the underlying basis of the interference effects were not described. Here, we show that targeting Nm formation to several individual sensitive sites in the peptidyl transferase center (PTC) strongly impairs ribosome accumulation and activity. Methylation was detected for all sites targeted, suggesting that the non-natural modification is the basis of the interference effects. For certain sensitive sites, the translation rate was reduced by 70-100%, due to: 1) a marked decrease (28-50%) in ribosomal subunits caused by slower pre-rRNA processing and mainly faster rRNA turn over and, 2) impaired activity of the surviving ribosomes. This last finding infers that the mis-targeted methylations compromise PTC function. The discovery that a new methylation can trigger robust rRNA degradation indicates that modification effects are monitored for quality control. These findings infer that nucleotide modifications can serve as evolutionary constraints and that snoRNPs mutations expected to occur in nature can cause human disease.

Mechanism of HIV-1 RNA dimerization in the central region of the genome, and significance for viral evolution

Background: HIV-1 genome dimerization is important for viral survival. Results: The central region of HIV-1 genome contains a G-rich sequence promoting recombination and capable of forming a dimeric G-quartet. Conclusion: Parallel alignment and proximity of two HIV-1 genomes associated at the 5′ end can be maintained through dimeric G-quartet formed in the central region of viral genome. Significance: Determining mechanism and factors involved in HIV-1 genome dimerization. The genome of HIV-1 consists of two identical or nearly identical RNA molecules. The RNA genomes are held in the same, parallel orientation by interactions at the dimer initiation site (DIS). Previous studies showed that in addition to interactions at DIS, sequences located 100 nucleotides downstream from the 5′ splice site can dimerize in vitro through an intermolecular G-quartet structure. Here we report that the highly conserved G-rich sequence in the middle portion of the HIV-1 genome near the central polypurine tract (cPPT) dimerizes spontaneously under high ionic strength in the absence of protein. The antisense RNA does not dimerize, strongly indicating that RNA dimerization does not exclusively involve A:U and G:C base pairing. The cation-dependent reverse transcriptase pausing profile, CD spectra profile, and cation-dependent association and thermal dissociation characteristics indicate G-quartet structures. Different forms of G-quartets are formed including monomers and, significantly, intermolecular dimers. Our results indicate that RNA genome dimerization and parallel alignment initiated through interactions at DIS may be greatly expanded and stabilized by formation of an intermolecular G-quartet at a distant site near the cPPT. It is likely that formation of G-quartet structure near the cPPT in vivo keeps the RNA genomes in proximity over a long range, promoting genetic recombination in numerous hot spots.

A sequence similar to tRNA3Lys gene is embedded in HIV-1 U3/R and promotes minus strand transfer

We identified a sequence embedded in the U3–R region of HIV-1 RNA that is highly complementary to human tRNA3Lys. The free energy of annealing to tRNA3Lys is significantly lower for this sequence and the primer-binding site than for other viral sequences of similar length. The only interruption in complementarity is a 29-nucleotide segment inserted where a tRNA intron would be expected. The insert contains the TATA box for viral RNA transcription. The embedded sequence includes a 9-nucleotide segment previously reported to aid minus-strand transfer by binding the primer tRNA3Lys. Reconstituting transfer in vitro, we show that including segments from the embedded sequence in the acceptor template, beyond the 9 nucleotides, further increases transfer efficiency. We propose that a gene encoding tRNA3Lys was incorporated during HIV-1 evolution and retained, largely intact, because of its roles in transcription and strand transfer.

Requirements for efficient minus strand strong-stop DNA transfer in human immunodeficiency virus 1

After HIV-1 enters a human cell, its RNA genome is converted into double stranded DNA during the multistep process of reverse transcription. First (minus) strand DNA synthesis is initiated near the 5’ end of the viral RNA, where only a short fragment of the genome is copied. In order to continue DNA synthesis the virus employs a complicated mechanism, which enables transferring of the growing minus strand DNA to a remote position at the genomic 3’ end. This is called minus strand DNA transfer. The transfer enables regeneration of long terminal repeat sequences, which are crucial for viral genomic DNA integration into the host chromosome. Numerous factors have been identified that stimulate minus strand DNA transfer. In this review we focus on describing protein-RNA and RNA-RNA interactions, as well as RNA structural features, known to facilitate this step in reverse transcription.

Deficiency in DNA damage response, a new characteristic of cells infected with latent HIV-1

ABSTRACT Viruses can interact with host cell molecules responsible for the recognition and repair of DNA lesions, resulting in dysfunctional DNA damage response (DDR). Cells with inefficient DDR are more vulnerable to therapeutic approaches that target DDR, thereby raising DNA damage to a threshold that triggers apoptosis. Here, we demonstrate that 2 Jurkat-derived cell lines with incorporated silent HIV-1 provirus show increases in DDR signaling that responds to formation of double strand DNA breaks (DSBs). We found that phosphorylation of histone H2AX on Ser139 (gamma-H2AX), a biomarker of DSBs, and phosphorylation of ATM at Ser1981, Chk2 at Thr68, and p53 at Ser15, part of signaling pathways associated with DSBs, are elevated in these cells. These results indicate a DDR defect even though the virus is latent. DDR-inducing agents, specifically high doses of nucleoside RT inhibitors (NRTIs), caused greater increases in gamma-H2AX levels in latently infected cells. Additionally, latently infected cells are more susceptible to long-term exposure to G-quadruplex stabilizing agents, and this effect is enhanced when the agent is combined with an inhibitor targeting DNA-PK, which is crucial for DSB repair and telomere maintenance. Moreover, exposing these cells to the cancer drug etoposide resulted in formation of DSBs at a higher rate than in un-infected cells. Similar effects of etoposide were also observed in population of primary memory T cells infected with latent HIV-1. Sensitivity to these agents highlights a unique vulnerability of latently infected cells, a new feature that could potentially be used in developing therapies to eliminate HIV-1 reservoirs.

The U1 snRNA hairpin II as a RNA affinity tag for selecting snoRNP complexes.

When isolating ribonucleoprotein (RNP) complexes by an affinity selection approach, tagging the RNA component can prove to be strategically important. This is especially true for purifying single types of snoRNPs, because in most cases the snoRNA is thought to be the only unique component. Here, we present a general strategy for selecting specific snoRNPs that features a high-affinity tag in the snoRNA and another in a snoRNP core protein. The RNA tag (called U1hpII) is a small (26 nt) stem-loop domain from human U1 snRNA. This structure binds with high affinity (K(D)=10(-11)M) to the RRM domain of the snRNP protein U1A. In our approach, the U1A protein contains a unique affinity tag and is coexpressed in vivo with the tagged snoRNA to yield snoRNP-U1A complexes with two unique protein tags-one in the bound U1A protein and the other in the snoRNP core protein. This scheme has been used effectively to select C/D and H/ACA snoRNPs, including both processing and modifying snoRNPs, and the snoRNA and core proteins are highly enriched. Depending on selection stringency other proteins are isolated as well, including an RNA helicase involved in snoRNP release from pre-rRNA and additional proteins that function in ribosome biogenesis. Tagging the snoRNA component alone is also effective when U1A is expressed with a myc-Tev-protein A fusion sequence. Combined with reduced stringency, enrichment of the U14 snoRNP with this latter system revealed potential interactions with two other snoRNPs, including one processing snoRNP involved in the same cleavages of pre-rRNA.

Sonic Hedgehog mimetic prevents leukocyte infiltration into the CNS during acute HIV infection

Infiltration of infected leukocytes culminates in establishment of a brain niche for Human Immunodeficiency Virus (HIV) during acute phase of infection, initiating an ongoing cascade of persistent viral replication and inflammation, that causes irreversible neuronal injury and HIV associated neurocognitive disease (HAND). In this study, humanized mice were treated with Smoothened Agonist (SAG), a Sonic Hedgehog (Shh) mimetic in order to fortify blood brain barrier (BBB) and dampen leukocyte extravasation into CNS during AHI. Results indicate that SAG treatment reduced viral burden in the CNS immediately after HIV transmission, but also conferred extended neuroprotection via increased BBB integrity (elevated levels of tight-junction protein, Claudin 5, and reduced S100B levels in periphery). These mice also showed healthier neurons with thick, uniform dendrites and reduced numbers of activated astrocytes. Additional in vitro experiments suggested SAG treatment was not associated with the establishment or reversal of latency in the target cells. Altogether, these findings validate neuroprotective role of Shh signaling and highlight the therapeutic potential of Shh mimetics against CNS complications associated with HIV infection. Further our results strongly demonstrate that pharmacological interventions to reduce leukocyte mobilization during early HIV infection, can provide prolonged neuroprotection, which might significantly delay the onset of HAND.