The 5′ m7G cap is an evolutionarily conserved modification of eukaryotic mRNA. Decades of research have established that the m7G cap serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export and cap-dependent protein synthesis. Only recently has the role of the cap 2′O methylation as an identifier of self RNA in the innate immune system against foreign RNA has become clear. The discovery of the cytoplasmic capping machinery suggests a novel level of control network. These new findings underscore the importance of a proper cap structure in the synthesis of functional messenger RNA. In this review, we will summarize the current knowledge of the biological roles of mRNA caps in eukaryotic cells. We will also discuss different means that viruses and their host cells use to cap their RNA and the application of these capping machineries to synthesize functional mRNA. Novel applications of RNA capping enzymes in the discovery of new RNA species and sequencing the microbiome transcriptome will also be discussed. We will end with a summary of novel findings in RNA capping and the questions these findings pose.

RNA interference (RNAi) has become a research tool to control gene expression in various organisms and holds potential as a new therapeutic strategy. The mechanism of small interfering RNA (siRNA)-mediated RNAi involves target mRNA cleavage and destruction in the cytoplasm. We investigated siRNA-mediated induction of RNAi in the nucleus of human cells. Notably, we observed highly efficient knockdown of small nuclear RNA 7SK by siRNA. siRNA- and microRNA-programmed RNA-induced silencing complexes (RISCs) were present in both cytoplasmic and nuclear compartments and specifically cleaved their perfectly matched target RNA with markedly high efficiencies. Our results provide the first evidence that human RISCs programmed with siRNA are present in the nucleus and can knock down target RNA levels. These studies reveal new roles for the RNAi machinery in modulating post-transcriptional gene expression in the nucleus.

The basis for the endothelial cell-restricted expression of endothelial nitric-oxide synthase (eNOS) is not known. While transgenic promoter/reporter mice demonstrated endothelium cell-specific eNOS expression, we found robust expression of episomal eNOS promoter/reporter constructs in cell types that do not express the native eNOS transcript. To explore the mechanism underlying this differential activity pattern of chromatin-versus episomebased eNOS promoters, we examined the methylation status of 5-regulatory sequences of the human eNOS gene. DNA methylation differed dramatically between endothelial and nonendothelial cell types, including vascular smooth muscle cells. This same cell type-specific methylation pattern was observed in vivo in endothelial and vascular smooth muscle cells of the mouse aorta at the native murine eNOS promoter. We addressed the functional consequences of methylation on eNOS transcription using transient transfection of in vitro methylated promoter/reporter constructs and found that methylated constructs exhibited a marked decrease in the synergistic action of Sp1, Sp3, and Ets1 on eNOS promoter activity. The addition of methyl-CpG-binding protein 2 further reduced the transcriptional activity of methylated eNOS constructs. Importantly, chromatin immunoprecipitation demonstrated the presence of Sp1, Sp3, and Ets1 at the native eNOS promoter in endothelial cells but not in vascular smooth muscle cells. Finally, robust expression of eNOS mRNA was induced in nonendothelial cell types following inhibition of DNA methyltransferase activity with 5-azacytidine, demonstrating the importance of DNA methylation-mediated repression. This report is the first to show that promoter DNA methylation plays an important role in the cell-specific expression of a constitutively expressed gene in the vascular endothelium.

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RNAi is a conserved mechanism in which small interfering RNAs (siRNAs) guide the degradation of cognate RNAs, but also promote heterochromatin assembly at repetitive DNA elements such as centromeric repeats1,2. However, the full extent of RNAi functions and its endogenous targets have not been explored. Here we show that in the fission yeast Schizosaccharomyces pombe, RNAi and heterochromatin factors cooperate to silence diverse loci, including sexual differentiation genes, genes encoding transmembrane proteins, and retrotransposons that are also targeted by the exosome RNA degradation machinery. In the absence of the exosome, transcripts are processed preferentially by the RNAi machinery, revealing siRNA clusters and corresponding increase in heterochromatin modifications across large domains containing genes and retrotransposons. We show that the generation of siRNAs and heterochromatin assembly by RNAi is triggered by a mechanism involving the canonical poly(A) polymerase Pla1 and an associated RNA surveillance factor Red1, which also activate the exosome. Remarkably, siRNA production and heterochromatin modifications at these target loci are regulated by environmental growth conditions, and by developmental signals that induce gene expression during sexual differentiation. Our analyses uncover interplay between RNAi and the exosome that is conserved in higher eukaryotes, and show that differentiation signals modulate RNAi silencing to regulate developmental genes.