Epstein-Barr virus-encoded EBNA2 alters immune checkpoint PD-L1 expression by downregulating miR-34a in B-cell lymphomas.

Cancer cells subvert host immune surveillance by altering immune checkpoint (IC) proteins. Some Epstein-Barr virus (EBV)-associated tumors have higher Programmed Cell Death Ligand, PD-L1 expression. However, it is not known how EBV alters ICs in the context of its preferred host, the B lymphocyte and in derived lymphomas. Here, we found that latency III-expressing Burkitt lymphoma (BL), diffuse large B-cell lymphomas (DLBCL) or their EBNA2-transfected derivatives express high PD-L1. In a DLBCL model, EBNA2 but not LMP1 is sufficient to induce PD-L1. Latency III-expressing DLBCL biopsies showed high levels of PD-L1. The PD-L1 targeting oncosuppressor microRNA miR-34a was downregulated in EBNA2-transfected lymphoma cells. We identified early B-cell factor 1 (EBF1) as a repressor of miR-34a transcription. Short hairpin RNA (shRNA)-mediated knockdown of EBF1 was sufficient to induce miR-34a transcription, which in turn reduced PD-L1. MiR-34a reconstitution in EBNA2-transfected DLBCL reduced PD-L1 expression and increased its immunogenicity in mixed lymphocyte reactions (MLR) and in three-dimensional biomimetic microfluidic chips. Given the importance of PD-L1 inhibition in immunotherapy and miR-34a dysregulation in cancers, our findings may have important implications for combinatorial immunotherapy, which include IC inhibiting antibodies and miR-34a, for EBV-associated cancers.

Human nuclear RNAi-defective 2 (NRDE2) is an essential RNA splicing factor.

The accurate inheritance of genetic material is a basic necessity in all domains of life and an unexpectedly large number of RNA processing factors are required for mitotic progression and genome stability. NRDE2 (nuclear RNAi defective-2) is an evolutionarily conserved protein originally discovered for its role in nuclear RNA interference (RNAi) and heritable gene silencing in Caenorhabditis elegans (C. elegans). The function of the human NRDE2 gene remains poorly understood. Here we show that human NRDE2 is an essential protein required for suppressing intron retention in a subset of pre-mRNAs containing short, GC-rich introns with relatively weak 5' and 3' splice sites. NRDE2 preferentially interacts with components of the U5 small nuclear ribonucleoprotein (snRNP), the exon junction complex, and the RNA exosome. Interestingly, NRDE2-depleted cells exhibit greatly increased levels of genomic instability and DNA damage, as well as defects in centrosome maturation and mitotic progression. We identify the essential centriolar satellite protein, CEP131, as a direct NRDE2-regulated target. NRDE2 specifically binds to and promotes the efficient splicing of CEP131 pre-mRNA, and depleting NRDE2 dramatically reduces CEP131 protein expression, contributing to impaired recruitment of critical centrosomal proteins (e.g., γ-tubulin and Aurora Kinase A) to the spindle poles during mitosis. Our work establishes a conserved role for human NRDE2 in RNA splicing, characterizes the severe genomic instability phenotypes observed upon loss of NRDE2, and highlights the direct regulation of CEP131 splicing as one of multiple mechanisms through which such phenotypes might be explained.

lin-4 and the NRDE pathway are required to activate a transgenic lin-4 reporter but not the endogenous lin-4 locus in C. elegans.

As the founding member of the microRNA (miRNA) gene family, insights into lin-4 regulation and function have laid a conceptual foundation for countless miRNA-related studies that followed. We previously showed that a transcriptional lin-4 reporter in C. elegans was positively regulated by a lin-4-complementary element (LCE), and by lin-4 itself. In this study, we sought to (1) identify additional factors required for lin-4 reporter expression, and (2) validate the endogenous relevance of a potential positive autoregulatory mechanism of lin-4 expression. We report that all four core nuclear RNAi factors (nrde-1, nrde-2, nrde-3 and nrde-4), positively regulate lin-4 reporter expression. In contrast, endogenous lin-4 levels were largely unaffected in nrde-2;nrde-3 mutants. Further, an endogenous LCE deletion generated by CRISPR-Cas9 revealed that the LCE was also not necessary for the activity of the endogenous lin-4 promoter. Finally, mutations in mature lin-4 did not reduce primary lin-4 transcript levels. Taken together, these data indicate that under growth conditions that reveal effects at the transgenic locus, a direct, positive autoregulatory mechanism of lin-4 expression does not occur in the context of the endogenous lin-4 locus.

Autoregulation of lin-4 microRNA transcription by RNA activation (RNAa) in C. elegans.

The conserved lin-4 microRNA (miRNA) regulates the proper timing of stem cell fate decisions in C. elegans by regulating stemness genes such as lin-14 and lin-28. (1)(-) (3) While lin-4 is upregulated toward the end of the first larval stage and functions as an essential developmental timing "switch", little is known about how lin-4 expression is regulated. (4) Here we show that in C. elegans hypodermal seam cells, transcription of lin-4 is positively regulated by lin-4 itself. In these cells, lin-4 activates its own transcription through a conserved lin-4-complementary element (LCE) in its promoter. We further show that lin-4 is required to recruit RNA polymerase II to its own promoter, and that lin-4 overexpression is sufficient for autoactivation. Finally, we show that a protein complex specifically binds the LCE in vitro, and that mutations that abolish this binding also reduce the in vivo expression of a plin-4:GFP reporter. Thus, we describe the first in vivo evidence of RNA activation (RNAa) by an endogenous miRNA, and provide new insights into an elegant autoregulatory mechanism that ensures the proper timing of stem cell fate decisions in development.

RNA-mediated gene activation.

The regulation of gene expression by non-coding RNAs (ncRNAs) has become a new paradigm in biology. RNA-mediated gene silencing pathways have been studied extensively, revealing diverse epigenetic and posttranscriptional mechanisms. In contrast, the roles of ncRNAs in activating gene expression remains poorly understood. In this review, we summarize the current knowledge of gene activation by small RNAs, long non-coding RNAs, and enhancer-derived RNAs, with an emphasis on epigenetic mechanisms.