Abstract
Gene expression in eukaryotes is dependent on the mRNA methyl cap which mediates mRNA processing and translation initiation. Synthesis of the methyl cap initiates with the addition of 7-methylguanosine to the initiating nucleotide of RNA pol II (polymerase II) transcripts, which occurs predominantly during transcription and in mammals is catalysed by RNGTT (RNA guanylyltransferase and 5′ phosphatase) and RNMT (RNA guanine-7 methyltransferase). RNMT has a methyltransferase domain and an N-terminal domain whose function is unclear; it is conserved in mammals, but not required for cap methyltransferase activity. In the present study we report that the N-terminal domain is necessary and sufficient for RNMT recruitment to transcription initiation sites and that recruitment occurs in a DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole)-dependent manner. The RNMT-activating subunit, RAM (RNMT-activating miniprotein), is also recruited to transcription initiation sites via an interaction with RNMT. The RNMT N-terminal domain is required for transcript expression, translation and cell proliferation.
Highlights
The methyl cap added to the 5 -end of RNA pol polymerase II (II) transcripts is required for eukaryotic gene expression
HeLa cells and IMECs were infected with retroviral constructs according to Abbreviations used: CTD, C-terminal domain; DMEM, Dulbecco’s modified Eagle’s medium; DRB, 5,6-dichloro-1-β-D-ribofuranosyl benzimidazole; GST, glutathione transferase; HA, haemagglutinin; IMEC, immortalized mammary epithelial cell; HEK, human embryonic kidney; NLS, nuclear localization signal; pol II, polymerase II; RAM, RNMT-activating miniprotein; RNA guanylyltransferase and phosphatase (RNGTT), RNA guanylyltransferase and 5 phosphatase; RNMT, RNA guanine-7 methyltransferase; TSS, transcriptional start site; WT, wild-type
Formation of the mRNA methyl cap occurs predominantly whilst transcripts are being synthesized, when RNGTT and RNMT are recruited to the transcribing RNA pol II [15,17,18]
Summary
The methyl cap added to the 5 -end of RNA pol II transcripts is required for eukaryotic gene expression. This structure protects mRNA from exonucleases, recruits the protein complexes which mediate RNA-processing events and forms a docking site for the eIF4F (eukaryotic initiation factor 4F) complex to initiate cap-dependent translation [1,2,3]. The methyl cap is created by the addition of a 7-methylguanosine group to the 5 -end of nascent transcripts, plus methylation of the initial transcribed nucleotides. RNMT (RNA guanine-7 methyltransferase) methylates the guanosine cap on the N-7 position to create the structure m7G(5 )ppp(5 )X [4]. Subsequent methylation of the transcribed nucleotides is catalysed by additional methyltransferases
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