Trimethylguanosine capping selectively promotes expression of Rev-dependent HIV-1 RNAs

Abstract

5′-mRNA capping is an early modification that affects pre-mRNA synthesis/splicing, RNA cytoplasmic transport, and mRNA translation and turnover. In eukaryotes, a 7-methylguanosine (m7G) cap is added to newly transcribed RNA polymerase II (RNAP II) transcripts. A subset of RNAP II-transcribed cellular RNAs, including small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and telomerase RNA, is further hypermethylated at the exocyclic N2 of the guanosine to create a trimethylguanosine (TMG)-capped RNA. Some of these TMG-capped RNAs are transported within the nucleus and from the nucleus to the cytoplasm by the CRM-1 (required for chromosome region maintenance) protein. CRM-1 is also used to export Rev/RRE-dependent unspliced/ partially spliced HIV-1 RNAs. Here we report that like snRNAs and snoRNAs, some Rev/RRE-dependent HIV-1 RNAs are TMG-capped. The methyltransferase responsible for TMG modification of HIV-1 RNAs is the human PIMT (peroxisome proliferator-activated receptor-interacting protein with methyltransferase) protein. TMG capping of unspliced/partially spliced HIV-1 RNAs represents a new regulatory mechanism for selective expression.required for chromosome region maintenance | RNA export | peroxisome proliferator-activated receptor-interacting protein with methyltransferase P osttranscriptional processing of RNA plays a critical role in gene expression (1-3). An early mRNA modification is the formation of a 7-methylguanosine (m7G) cap (4-6). In mammals, the acquisition of an m7G cap requires two enzymes (7-9), a capping enzyme (triphosphatase and guanylyltransferase activity) and an RNA-(guanine 7)-methyltransferase. These proteins are essential for cell growth, and mutations in the triphosphatase, guanylyltransferase, or methyltransferase components of the capping apparatus are lethal in vivo (10).Cellular RNAP II transcripts are mostly m7G-capped (1-3, 11). An m7G cap facilitates the initiation of translation in mammalian cells, and a failure to cap premRNAs results in their accelerated decay by 5′ exoribonuclease degradation (2,(12)(13)(14). Capping of viral RNAs has been less extensively investigated. It has been generally assumed that like cellular RNAs, many viral RNAs have a 5′ m7G cap, and that viruses either use the cellular capping machinery (e.g., viruses that replicate in the nucleus) or encode their own capping enzymes (e.g., viruses that replicate in the cytoplasm) (15, 16). Interestingly, there is a surprisingly diverse range of cap modifications; for instance, Mimivirus, a large DNA virus of amoeba, encodes an RNA cap guanine-N2 methyltransferase that hypermethylates the viral RNA cap (17); influenza virus "snatches" caps from cellular mRNAs (18, 19); West Nile fever virus has two methyl additions on its RNA cap (20); Sindbis virus produces mRNAs that have dimethylguanosine and trimethylguanosine caps [hypermethylated caps/m 2,2,7 G caps (21)]; and Semliki forest virus late mRNAs also have hypermethylated caps (22). On the other hand, poliovirus, encephalomyelitis virus, foot and mouth...

An enzyme-coupled high-throughput assay for screening RNA methyltransferase activity inE. Colicell lysate

“…As a proof of concept, we tested three known methyltransferase inhibitors and found that only (-)-epigallocatechin gallate inhibited hTGS1. Since specific RNA methyltransferases inhibitors, e.g., for Dengue virus methyltransferase 29 or even for PIMT 30 could be of therapeutic interest, a high-throughput screen is desirable to test large compound libraries.…”

“…29 Even some HIV-1 RNAs have been found to be trimethylguanosine-capped and acquisition of this cap hypermethylations facilitates the optimal expression of these RNAs. 30 An assay to test potential small molecule inhibitors of RNA methyltransferases including trimethylguanosine synthases in high-throughput could thus speed up the discovery of novel inhibitors.…”

An Exportin-1–dependent microRNA biogenesis pathway during human cell quiescence

“…Interestingly, Exportin-1 also has high affinity for the (TMG)-capped small nucleolar RNA (snoRNA) U3 in the nucleus and transports it from Cajal bodies to the nucleoli (32). Another study showed that TGS1 enhances Rev-dependent HIV-1 RNA expression by (TMG)-capping viral mRNAs in the nucleus, thereby increasing recognition by Exportin-1 for transport to the cytoplasm (33).…”

Translation initiation of the HIV-1 mRNA

“…However, this is not the case and TMG-capping of viral mRNAs associates with an increased expression of HIV-1 structural proteins. 151 Although the mechanism by which these TMG-capped viral mRNAs recruit the translational machinery was not studied, it is tempting to speculate that translation initiation from the TMG-HIV-1 RNAs could take place by a cap-independent IRES-mediated mechanism.…”

“…PIMT hypermethylates the m 7 G RNA-cap to generate a pool of trimethylguanosine (TMG; m 2, 2,7 guanosine)-cap containing viral mRNAs. [151][152][153] TMG-capped-RNAs are known to translate poorly, 154 due to the inefficient recognition of the trimethyl by the cap-binding protein eIF4E. [155][156][157][158] As a consequence, it would be predicted that TMG-HIV-1 RNAs would be inefficiently translated resulting in a decrease of viral expression.…”