Enhancer-promoter activation by the Kaposi sarcoma-associated herpesvirus episome maintenance protein LANA.

Higher-order genome structure influences the transcriptional regulation of cellular genes through the juxtaposition of regulatory elements, such as enhancers, close to promoters of target genes. While enhancer activation has emerged as an important facet of Kaposi sarcoma-associated herpesvirus (KSHV) biology, the mechanisms controlling enhancer-target gene expression remain obscure. Here, we discover that the KSHV genome tethering protein latency-associated nuclear antigen (LANA) potentiates enhancer-target gene expression in primary effusion lymphoma (PEL), a highly aggressive B cell lymphoma causally associated with KSHV. Genome-wide analyses demonstrate increased levels of enhancer RNA transcription as well as activating chromatin marks at LANA-bound enhancers. 3D genome conformation analyses identified genes critical for latency and tumorigenesis as targets of LANA-occupied enhancers, and LANA depletion results in their downregulation. These findings reveal a mechanism in enhancer-gene coordination and describe a role through which the main KSHV tethering protein regulates essential gene expression in PEL.

RNA recognition by PKR during DNA virus infection.

Protein kinase R (PKR) is a double-stranded RNA (dsRNA) binding protein that plays a crucial role in innate immunity during viral infection and can restrict both DNA and RNA viruses. The potency of its antiviral function is further reflected by the large number of viral-encoded PKR antagonists. However, much about the regulation of dsRNA accumulation and PKR activation during viral infection remains unknown. Since DNA viruses do not have an RNA genome or RNA replication intermediates like RNA viruses do, PKR-mediated dsRNA detection in the context of DNA virus infection is particularly intriguing. Here, we review the current state of knowledge regarding the regulation of PKR activation and its antagonism during infection with DNA viruses.

5-Fluorouracil Treatment Represses Pseudouridine-Containing Small RNA Export into Extracellular Vesicles.

5-fluorouracil (5-FU) has been used for chemotherapy for colorectal and other cancers for over 50 years. The prevailing view of its mechanism of action is inhibition of thymidine synthase leading to defects in DNA replication and repair. However, 5-FU is also incorporated into RNA causing toxicity due to defects in RNA metabolism, inhibition of pseudouridine modification, and altered ribosome function. Here, we examine the impact of 5-FU on the expression and export of small RNAs (sRNAs) into small extracellular vesicles (sEVs). Moreover, we assess the role of 5-FU in regulation of post-transcriptional sRNA modifications (PTxM) using mass spectrometry approaches. EVs are secreted by all cells and contain a variety of proteins and RNAs that can function in cell-cell communication. PTxMs on cellular and extracellular sRNAs provide yet another layer of gene regulation. We found that treatment of the colorectal cancer (CRC) cell line DLD-1 with 5-FU led to surprising differential export of miRNA snRNA, and snoRNA transcripts. Strikingly, 5-FU treatment significantly decreased the levels of pseudouridine on both cellular and secreted EV sRNAs. In contrast, 5-FU exposure led to increased levels of cellular sRNAs containing a variety of methyl-modified bases. Our results suggest that 5-FU exposure leads to altered expression, base modifications, and mislocalization of EV base-modified sRNAs.