Evaluation of the RNA-dependence of PRC2 binding to chromatin in human pluripotent stem cells.

Polycomb Repressive Complex 2 (PRC2), an important histone modifier and epigenetic repressor, has been known to interact with RNA for almost two decades. In our previous publication (Long, Hwang et al. 2020), we presented data supporting the functional importance of RNA interaction in maintaining PRC2 occupancy on chromatin, using comprehensive approaches including an RNA-binding mutant of PRC2 and an rChIP-seq assay. Recently, concerns have been expressed regarding whether the RNA-binding mutant has impaired histone methyltransferase activity and whether the rChIP-seq assay can potentially generate artifacts. Here we provide new data that support a number of our original findings. First, we found the RNA-binding mutant to be fully capable of maintaining H3K27me3 levels in human induced pluripotent stem cells. The mutant had reduced methyltransferase activity in vitro, but only on some substrates at early time points. Second, we found that our rChIP-seq method gave consistent data across antibodies and cell lines. Third, we further optimized rChIP-seq by using lower concentrations of RNase A and incorporating a catalytically inactive mutant RNase A as a control, as well as using an alternative RNase (RNase T1). The EZH2 rChIP-seq results using the optimized protocols supported our original finding that RNA interaction contributes to the chromatin occupancy of PRC2.

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells.

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Two herpesviral noncoding PAN RNAs are functionally homologous but do not associate with common chromatin loci.

During lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear viral long noncoding RNA known as PAN RNA becomes the most abundant polyadenylated transcript in the cell. Knockout or knockdown of KSHV PAN RNA results in loss of late lytic viral gene expression and, consequently, reduction of progeny virion release from the cell. Here, we demonstrate that knockdown of PAN RNA from the related Rhesus macaque rhadinovirus (RRV) phenocopies that of KSHV PAN RNA. These two PAN RNA homologs, although lacking significant nucleotide sequence conservation, can functionally substitute for each other to rescue phenotypes associated with the absence of PAN RNA expression. Because PAN RNA is exclusively nuclear, previous studies suggested that it directly interacts with host and viral chromatin to modulate gene expression. We studied KSHV and RRV PAN RNA homologs using capture hybridization analysis of RNA targets (CHART) and observed their association with host chromatin, but the loci differ between PAN RNA homologs. Accordingly, we find that KSHV PAN RNA is undetectable in chromatin following cell fractionation. Thus, modulation of gene expression at specific chromatin loci appears not to be the primary, nor the pertinent function of this viral long noncoding RNA. PAN RNA represents a cautionary tale for the investigation of RNA association with chromatin whereby cross-linking of DNA spatially adjacent to an abundant nuclear RNA gives the appearance of specific interactions. Similarly, PAN RNA expression does not affect viral transcription factor complex expression or activity, which is required for generation of the late lytic viral mRNAs. Rather, we provide evidence for an alternative model of PAN RNA function whereby knockdown of KSHV or RRV PAN RNA results in compromised nuclear mRNA export thereby reducing the cytoplasmic levels of viral mRNAs available for production of late lytic viral proteins.

Kaposi's Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Foci Is Influenced by Viral DNA Replication and Viral Noncoding Polyadenylated Nuclear RNA.

Kaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, replicates within the nuclei of its human cell host and hijacks host machinery for expression of its genes. The activities that culminate in viral DNA synthesis and assembly of viral proteins into capsids physically concentrate in nuclear areas termed viral replication compartments. We sought to better understand the spatiotemporal regulation of viral RNAs during the KSHV lytic phase by examining and quantifying the subcellular localization of select viral transcripts. We found that viral mRNAs, as expected, localized to the cytoplasm throughout the lytic phase. However, dependent on active viral DNA replication, viral transcripts also accumulated in the nucleus, often in foci in and around replication compartments, independent of the host shutoff effect. Our data point to involvement of the viral long noncoding polyadenylated nuclear (PAN) RNA in the localization of an early, intronless viral mRNA encoding ORF59-58 to nuclear foci that are associated with replication compartments.IMPORTANCE Late in the lytic phase, mRNAs from Kaposi's sarcoma-associated herpesvirus accumulate in the host cell nucleus near viral replication compartments, centers of viral DNA synthesis and virion production. This work contributes spatiotemporal data on herpesviral mRNAs within the lytic host cell and suggests a mechanism for viral RNA accumulation. Our findings indicate that the mechanism is independent of the host shutoff effect and splicing but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, PAN RNA. PAN RNA is essential for the viral life cycle, and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus.

Mechanisms of Very Long Abortive Transcript Release during Promoter Escape.

A phage T5 N25 promoter variant, DG203, undergoes the escape transition at the +16 to +19 positions after transcription initiation. By specifically examining the abortive activity of the initial transcribing complex at position +19 (ITC19), we observe the production of both GreB-sensitive and GreB-resistant VLAT19. This suggests that ITC19, which is perched on the brink of escape, is highly unstable and can achieve stabilization through either backtracking or forward translocation. Of the forward-tracked fraction, only a small percentage escapes normally (followed by stepwise elongation) to produce full-length RNA; the rest presumably hypertranslocates to release GreB-resistant VLATs. VLAT formation is dependent not only on consensus -35/-10 promoters with 17 bp spacing but also on sequence characteristics of the spacer DNA. Analysis of DG203 promoter variants containing different spacer sequences reveals that AT-rich spacers intrinsically elevate the level of VLAT formation. The AT-rich spacer of DG203 joined to the -10 box presents an UP element sequence capable of interacting with the polymerase α subunit C-terminal domain (αCTD) during the escape transition, which in turn enhances VLAT release. Utilization of the spacer/-10 region UP element by αCTD subunits requires a 10-15 bp hypertranslocation. We document the physical occurrence of hyper forward translocation using ExoIII footprinting analysis.

Viral noncoding RNAs: more surprises.

Eukaryotic cells produce several classes of long and small noncoding RNA (ncRNA). Many DNA and RNA viruses synthesize their own ncRNAs. Like their host counterparts, viral ncRNAs associate with proteins that are essential for their stability, function, or both. Diverse biological roles--including the regulation of viral replication, viral persistence, host immune evasion, and cellular transformation--have been ascribed to viral ncRNAs. In this review, we focus on the multitude of functions played by ncRNAs produced by animal viruses. We also discuss their biogenesis and mechanisms of action.