Biology subjects

Bogerd, H. P.

Publications and source records attributed to Bogerd, H. P..

6 recordsLinked to original sources

bioRxiv2021

Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique

Pseudouridine ({Psi}) is the most common non-canonical ribonucleoside present on mammalian non-coding RNAs (ncRNAs), including rRNAs, tRNAs and snRNAs, where it contributes [~]7% of the total uridine level. However, {Psi} constitutes only [~]0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. {Psi} residues have been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of {Psi} residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based {Psi} mapping technique called photo-crosslinking assisted {Psi} sequencing (PA-{Psi}-seq) and use it to map {Psi} residues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe several 293T-derived cell lines in which human PUS enzymes previously reported to add {Psi} residues to human mRNAs, specifically PUS1, PUS7 and TRUB1/PUS4, were inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of {Psi} addition on cellular mRNAs to each of these three PUS enzymes, the {Psi} sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7 or TRUB1 function did not significantly reduce the level of {Psi} residues detected on total human mRNA below the [~]0.1% level seen in wild type cells, thus implying that the PUS enzyme(s) that adds the bulk of {Psi} residues to human mRNAs remains to be defined.

molecular biology↗

bioRxiv2021

Epitranscriptomic addition of m6A regulates HIV-1 RNA stability and alternative splicing

Previous work in several laboratories has demonstrated that the epitranscriptomic addition of m6A to viral transcripts promotes the replication and pathogenicity of a wide range of DNA and RNA viruses, yet the underlying mechanisms responsible for this positive effect have remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, yet how m6A readers regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m6A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is in large part mediated by the the nuclear m6A reader YTHDC1 and the cytoplasmic m6A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs while YTHDF2 binding correlates with increased HIV-1 transcript stability. Author SummaryThis manuscript reports that the expression of mRNAs encoded by the pathogenic human retrovirus HIV-1 is regulated by the methylation of a small number of specific adenosine residues. These in turn recruit a nuclear RNA binding protein, called YTHDC1, which modulates the alternative splicing of HIV-1 transcripts, as well as a cytoplasmic RNA binding protein, called YTHDF2, which stabilizes viral mRNAs. The regulation of HIV-1 gene expression by adenosine methylation is therefore critical for the effective and ordered expression of HIV-1 mRNAs and could represent a novel target for antiviral development.

molecular biology↗

bioRxiv2020

Revealing the high propensity of RNAs to non-specifically bind drug-like small molecules

Identifying small molecules that selectively bind a single RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to non-specific binding of aminoglycosides and intercalators to a variety of RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities, however the ability of such compounds to discriminate against RNA stem-loops commonly found in the transcriptome has not been thoroughly assessed in all cases. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activity of three distinct RNAs, to non-specifically bind two HIV-1 stem-loop RNAs: the transactivation response element (TAR) and stem IIB in the rev response element (RREIIB). All three compounds bound to TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting substantial off-target interactions consistent with non-specific cellular activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, much like aminoglycosides, and in contrast to ligands that specifically bind riboswitches. Our results support extending the group of non-selective RNA-binders beyond aminoglycosides and intercalators to encompass drug-like compounds with capacity for non-specific hydrogen-bonding and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.

biochemistry↗

bioRxiv2020

Robust HIV-1 Replication in the Absence of Integrase Function.

Integration of the proviral DNA intermediate into the host cell genome represents an essential step in the retroviral life cycle. While the reason(s) for this requirement remains unclear, it is known that unintegrated proviral DNA is epigenetically silenced. Here, we demonstrate that HIV-1 mutants lacking functional integrase can mount a robust, spreading infection in cells expressing the Tax transcription factor encoded by human T-cell leukemia virus 1. In these cells, HIV-1 forms episomal DNA circles, analogous to Hepatitis B virus covalently closed circular DNAs (cccDNAs), that are transcriptionally active and fully capable of supporting viral replication. This rescue correlates with the loss of inhibitory epigenetic marks, and the acquisition of activating marks, on histones bound to unintegrated HIV-1 DNA. Thus retroviral DNA integration may have evolved, at least in part, as a mechanism to avoid the epigenetic silencing of extrachromosomal viral DNA by host innate antiviral factors. SignificanceWhile retroviral DNA is synthesized normally after infection by integrase-deficient viruses, the resultant episomal DNA is then epigenetically silenced. Here, we show that expression of the Tax transcription factor encoded by a second human retrovirus, HTLV-1, prevents the epigenetic silencing of unintegrated HIV-1 DNA and instead induces the addition of activating epigenetic marks, and the recruitment of NF-kB/Rel proteins, to the HIV-1 LTR promoter. Moreover, in the presence of Tax, the HIV-1 DNA circles that form in the absence of integrase function are not only efficiently transcribed but also support a spreading, pathogenic IN- HIV-1 infection. Thus, retroviruses have the potential to replicate without integration, as is indeed seen with HBV.

microbiology↗

bioRxiv2019

Stabilizing non-native excited states as a therapeutic strategy for targeting RNA

Many regulatory RNAs undergo changes in their structure from the dominant ground-state (GS) toward short-lived low-abundance excited-states (ES) that reorganize local elements of secondary structure. ESs are increasingly observed in vitro and implicated in the folding and biological activities of regulatory RNAs and as targets for developing therapeutics. However, whether these ESs also form with comparable abundance within the complex cellular environment remains unknown. Here, we developed an approach for assessing the relative stability and abundance of RNA ESs within the functional cellular context. The approach uses point substitution mutations to increase the population of an inactive ES relative to the active GS. The cellular activity of such ES-stabilizing mutants then provides an indirect measure of any residual population of the active GS within the functional cellular context. Compensatory rescue mutations that restore the GS are used to control for changes in cellular activity arising due to changes in sequence. The approach is applied to probe ESs in two highly conserved and functionally important regulatory RNAs from HIV-1: the transactivation response element (TAR) and the Rev response element (RRE). For both RNAs, ES-stabilizing mutations inhibited cellular activity to a degree that correlates with the extent to which they stabilize the ES relative to the GS in vitro. These results indicate that the non-native ESs of TAR and RRE likely form in cells with abundances comparable to those measured in vitro and their targeted stabilization provides a new avenue for developing anti-HIV therapeutics.

biochemistry↗

bioRxiv2019

Extensive epitranscriptomic methylation of A and C residues on murine leukemia virus transcripts enhances viral gene expression

While it has been known for several years that viral RNAs are subject to the addition of several distinct covalent modifications to individual nucleotides, collectively referred to as epitranscriptomic modifications, the effect of these editing events on viral gene expression has been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic RNA to homogeneity and show that this viral RNA contains levels of N6-methyladenosine (m6A), 5-methylcytosine (m5C) and 2O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of m6A and m5C residues on MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV variants bearing silent mutations that removed a subset of these sites. Analysis of the replication potential of these mutants revealed a modest but significant attenuation in viral replication in 3T3 cells in culture. Consistent with a positive role for m6A and m5C in viral replication, we also demonstrate that overexpression of the key m6A reader protein YTHDF2 enhances MLV replication, while downregulation of the m5C writer NSUN2 inhibits MLV replication.\n\nImportanceThe data presented in this manuscript demonstrate that MLV RNAs bear an exceptionally high level of the epitranscriptomic modifications m6A, m5C and Nm, thus suggesting that these each facilitate some aspect of the viral replication cycle. Consistent with this hypothesis, we demonstrate that mutational removal of a subset of these m6A or m5C modifications from MLV transcripts inhibits MLV replication in cis and a similar result was also observed upon manipulation of the level of expression of key cellular epitranscriptomic cofactors in trans. Together, these results argue that the addition of several different epitranscriptomic modifications to viral transcripts stimulates viral gene expression and suggest that MLV has therefore evolved to maximize the level of these modifications that are added to viral RNAs.

microbiology↗