To cap it all off, again: dynamic capping and recapping of coding and non-coding RNAs to control transcript fate and biological activity.

The addition of the methyl-7-guanosine (m7G) "cap" on the 5' ends of coding and some non-coding RNAs is essential for their protein coding capacity and biochemical activity, respectively. It was previously considered that capping was a constitutive process that generates a complete cap on all transcripts at steady-state. However, development of new methodologies demonstrated that steady-state capping is a dynamic and regulatable feature of many coding and non-coding RNAs. Indeed, capping status of specific RNAs can flux during differentiation and development, thereby impacting on their protein-coding capacity and activity. Moreover, in some primary cancer specimens, capping can be elevated for transcripts encoding proteins involved in proliferation and survival corresponding to their increased protein levels. Overexpression of one of the capping enzymes (RNMT), the transcription factor MYC or the eukaryotic translation initiation factor eIF4E all led to increased levels of steady-state capping of selected transcripts. Additionally, transcripts can be decapped and recapped, allowing these to be sequestered until needed. This review provides a summary of the major advances in enzymatic and affinity-based approaches to quantify m7G capping. Further, we summarize the evidence for regulation of capping. Capping has emerged as a significant regulatory step in RNA metabolism which is poised to impact a myriad of biological processes.

Human IFIT1 Inhibits mRNA Translation of Rubulaviruses but Not Other Members of the Paramyxoviridae Family.

UNLABELLED: We have previously shown that IFIT1 is primarily responsible for the antiviral action of interferon (IFN) alpha/beta against parainfluenza virus type 5 (PIV5), selectively inhibiting the translation of PIV5 mRNAs. Here we report that while PIV2, PIV5, and mumps virus (MuV) are sensitive to IFIT1, nonrubulavirus members of the paramyxoviridae such as PIV3, Sendai virus (SeV), and canine distemper virus (CDV) are resistant. The IFIT1 sensitivity of PIV5 was not rescued by coinfection with an IFIT1-resistant virus (PIV3), demonstrating that PIV3 does not specifically inhibit the antiviral activity of IFIT1 and that the inhibition of PIV5 mRNAs is regulated by cis-acting elements. We developed an in vitro translation system using purified human IFIT1 to further investigate the mechanism of action of IFIT1. While the translations of PIV2, PIV5, and MuV mRNAs were directly inhibited by IFIT1, the translations of PIV3, SeV, and CDV mRNAs were not. Using purified human mRNA-capping enzymes, we show biochemically that efficient inhibition by IFIT1 is dependent upon a 5' guanosine nucleoside cap (which need not be N7 methylated) and that this sensitivity is partly abrogated by 2'O methylation of the cap 1 ribose. Intriguingly, PIV5 M mRNA, in contrast to NP mRNA, remained sensitive to inhibition by IFIT1 following in vitro 2'O methylation, suggesting that other structural features of mRNAs may influence their sensitivity to IFIT1. Thus, surprisingly, the viral polymerases (which have 2'-O-methyltransferase activity) of rubulaviruses do not protect these viruses from inhibition by IFIT1. Possible biological consequences of this are discussed. IMPORTANCE: Paramyxoviruses cause a wide variety of diseases, and yet most of their genes encode structural proteins and proteins involved in their replication cycle. Thus, the amount of genetic information that determines the type of disease that paramyxoviruses cause is relatively small. One factor that will influence disease outcomes is how they interact with innate host cell defenses, including the interferon (IFN) system. Here we show that different paramyxoviruses interact in distinct ways with cells in a preexisting IFN-induced antiviral state. Strikingly, all the rubulaviruses tested were sensitive to the antiviral action of ISG56/IFIT1, while all the other paramyxoviruses tested were resistant. We developed novel in vitro biochemical assays to investigate the mechanism of action of IFIT1, demonstrating that the mRNAs of rubulaviruses can be directly inhibited by IFIT1 and that this is at least partially because their mRNAs are not correctly methylated.

Burkitt's lymphoma-associated c-Myc mutations converge on a dramatically altered target gene response and implicate Nol5a/Nop56 in oncogenesis.

Burkitt's lymphomas (BLs) acquire consistent point mutations in a conserved domain of Myc, Myc Box I. We report that the enhanced transforming activity of BL-associated Myc mutants can be uncoupled from loss of phosphorylation and increased protein stability. Furthermore, two different BL-associated Myc mutations induced similar gene expression profiles independently of T58 phosphorylation, and these profiles are dramatically different from MycWT. Nol5a/Nop56, which is required for ribosomal RNA methylation, was identified as a gene hyperactivated by the BL-associated Myc mutants. We show that Nol5a is necessary for Myc-induced cell transformation, enhances MycWT-induced cell transformation and increases the size of MycWT-induced tumors. Thus, Nol5a expands the link between Myc-induced regulation of nucleolar target genes, which are rate limiting for cell transformation and tumor growth.

Enhanced mRNA cap methylation increases cyclin D1 expression and promotes cell transformation.

Cap-dependent mRNA translation requires the methylation of the mRNA guanosine cap by RNA guanine-7-methyltransferase (RNMT). mRNA cap methylation was recently described to be rate-limiting for a subset of mRNAs, and to be enhanced by expression of c-Myc and E2F1, although the biological significance of this finding was not investigated. Here, it is reported that increased RNMT expression enhances cellular mRNA cap methyltransferase activity, promotes mammary epithelial cell transformation and cooperates with H-RasV12 or c-Myc to promote fibroblast cell transformation. Cyclin D1 is a prominent oncogene in epithelial tumours. A significant fraction of Cyclin D1 mRNA was found to be unmethylated on the mRNA cap and thus dormant in mammary epithelial cells. Cyclin D1 expression was increased by enhanced mRNA cap methylation. In summary, this report shows that mRNA cap methylation is rate-limiting for expression of an oncogene and cell transformation.

Specific regulation of mRNA cap methylation by the c-Myc and E2F1 transcription factors.

Methylation of the mRNA 5' guanosine cap is essential for efficient gene expression. The 5' methyl cap binds to eIF4E, which is the first step in the recruitment of mRNA to the 40S ribosomal subunit. To investigate whether mRNA cap methylation is regulated in a gene-specific manner, we established a method to detect the relative level of cap methylation on specific mRNAs. We found that two transcription factors, c-Myc and E2F1, induce cap methylation of their transcriptional target genes, and therefore, c-Myc and E2F1 upregulate gene expression by simultaneously inducing transcription and promoting translation. c-Myc-induced cap methylation is greater than transcriptional induction for the majority of its target genes, indicating that this is a major mechanism by which Myc regulates gene expression.

An N-Myc truncation analogous to c-Myc-S induces cell proliferation independently of transactivation but dependent on Myc homology box II.

Myc promotes both normal cell proliferation and oncogenic transformation through the activation and repression of target genes. The c-Myc-S protein is a truncated form of c-Myc that is produced in some cells from translation initiation at an internal AUG codon. We report that c-Myc-S and a similar truncated form of N-MycWT can fully rescue the proliferation defect in myc-null fibroblasts, but rescue is dependent on the highly conserved Myc homology box II (MBII). Global gene expression studies show that the N-Myc equivalent of c-Myc-S is defective for virtually all transcriptional activation of Myc target genes but remains active for the majority of transcriptional repression. Repression by Myc-S is dependent on MBII, but it does not bind to several known nuclear cofactors. These data suggest that repression by Myc involves recruitment of a novel MBII-dependent cofactor.

E-cadherin repression contributes to c-Myc-induced epithelial cell transformation.

c-Myc oncoprotein is overexpressed in a significant proportion of human epithelial cancers, and experimental overexpression of c-Myc in epithelial cells promotes tumour formation. However, it is not known how c-Myc promotes epithelial cell tumour formation. We report that c-Myc expression in human mammary epithelial cells induces a dramatic change in cell morphology, with some characteristics of an 'epithelial to mesenchymal transition'. E-cadherin expression is repressed by a post-transcriptional mechanism in cells expressing c-Myc. Furthermore, E-cadherin repression is necessary for c-Myc-induced cell transformation.