N⁶-methyladenosine (m⁶A) is the most abundant internal RNA modification of cellular mRNAs. m⁶A is recognised by YTH domain-containing proteins, which selectively bind to m⁶A-decorated RNAs regulating their turnover and translation. Using an m⁶A-modified hairpin present in the Kaposi’s sarcoma associated herpesvirus (KSHV) ORF50 RNA, we identified seven members from the ‘Royal family’ as putative m⁶A readers, including SND1. RIP-seq and eCLIP analysis characterised the SND1 binding profile transcriptome-wide, revealing SND1 as an m⁶A reader. We further demonstrate that the m⁶A modification of the ORF50 RNA is critical for SND1 binding, which in turn stabilises the ORF50 transcript. Importantly, SND1 depletion leads to inhibition of KSHV early gene expression showing that SND1 is essential for KSHV lytic replication. This work demonstrates that members of the ‘Royal family’ have m⁶A-reading ability, greatly increasing their epigenetic functions beyond protein methylation.

Despite the nuclear localization of the m⁶A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m⁶A-modified. However, these findings are mostly based on m⁶A-Seq, an antibody-dependent technique with a high rate of false positives. Here, we address the presence of m⁶A in CHIKV and DENV RNAs. For this, we combine m⁶A-Seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we find no evidence of m⁶A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m⁶A machinery does not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection has no effect on the m⁶A machinery’s localization. Our results challenge the prevailing notion that m⁶A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.

The epitranscriptomic modification N⁶-methyladenosine (m⁶A) is a ubiquitous feature of the mammalian transcriptome. It modulates mRNA fate and dynamics to exert regulatory control over numerous cellular processes and disease pathways, including viral infection. Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation from the latent phase leads to the redistribution of m⁶A topology upon both viral and cellular mRNAs within infected cells. Here we investigate the role of m⁶A in cellular transcripts upregulated during KSHV lytic replication. Our results show that m⁶A is crucial for the stability of the GPRC5A mRNA, whose expression is induced by the KSHV latent–lytic switch master regulator, the replication and transcription activator (RTA) protein. Moreover, we demonstrate that GPRC5A is essential for efficient KSHV lytic replication by directly regulating NFκB signalling. Overall, this work highlights the central importance of m⁶A in modulating cellular gene expression to influence viral infection.

Integrator cleaves nascent RNA, triggering RNA polymerase II transcription termination, but how cleavage is regulated is poorly understood. Here we show hnRNPUL1 ensures efficient Integrator-mediated cleavage of nascent RNA downstream of snRNA genes and, in the case of U2 snRNA, binds a terminal stem-loop involved in this process. In the nucleoplasm, hnRNPUL1 binds U4 snRNA and SART3 and enables efficient reformation of the U4:U6 di-snRNP for further rounds of pre-mRNA splicing. Sustained hnRNPUL1 loss leads to reduced levels of snRNAs, defects in histone mRNA 3′ end processing and loss of Cajal bodies. hnRNPUL1 binds RNA through multiple domains, including a globular central domain comprising tightly juxtaposed SPRY and dead polynucleotide kinase folds. This latter fold allows binding to 5′-monophosphorylated RNAs in a mutually exclusive manner with ATP binding and functions as an XRN2 antagonist when overexpressed. We identify a cohort of amyotrophic lateral sclerosis patients harbouring disruptive mutations in hnRNPUL1. SMN loss in spinal muscular atrophy and hnRNPUL1 loss both disrupt snRNP biogenesis, leading to motor neuron death, suggesting a common aetiology.

Despite the nuclear localization of the m ⁶ A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m ⁶ A-modified. However, these findings are mostly based on m ⁶ A-seq, an antibody-dependent technique with a high rate of false positives. Here, we addressed the presence of m ⁶ A in CHIKV and DENV RNAs. For this, we combined m ⁶ A-seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we found no evidence of m ⁶ A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m ⁶ A machinery did not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection had no effect on the m ⁶ A machinery’s localization. Our results challenge the prevailing notion that m ⁶ A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.

Identifying biomolecules that shape nuclear organization is essential for understanding gene regulation in health and disease. Oncogenic fusion proteins rewire chromosome folding and generate biomolecular condensates, but the cofactors of oncoprotein-driven chromatin regulation remain poorly defined, and whether such factors have analogous functions in fusion-naïve cells is unknown. We find that NSD3 mediates chromosome folding in fusion-positive and fusion-negative cells. NSD3 stabilizes the BRD4-NUT fusion oncoprotein on chromatin, promotes histone H3K36me2, and supports oncogene expression while maintaining BRD4-NUT nuclear condensates. NSD3 loss attenuates distant chromatin interactions between BRD4-NUT megadomains both within and between chromosomes. In cells lacking BRD4-NUT, the short, catalytically inactive isoform of NSD3, NSD3short, promotes chromatin contacts separated by multiple megabases. The ability of NSD3short to promote long-range chromatin contacts requires its PWWP domain. By combining chromatin structural analyses in fusion-positive and fusion-negative cells, we show that interrogating fusion oncoprotein-driven chromosome misfolding reveals the multicomponent basis of nuclear compartmentalization and uncovers an adaptor protein that promotes chromatin contacts independent of enzymatic activity.