Abstract
Eukaryotic gene expression is dependent on the modification of the first transcribed nucleotide of pre-mRNA by the addition of the 7-methylguanosine cap. The cap protects transcripts from exonucleases and recruits complexes which mediate transcription elongation, processing and translation initiation. The cap is synthesized by a series of reactions which link 7-methylguanosine to the first transcribed nucleotide via a 5′ to 5′ triphosphate bridge. In mammals, cap synthesis is catalysed by the sequential action of RNGTT (RNA guanylyltransferase and 5′-phosphatase) and RNMT (RNA guanine-7 methyltransferase), enzymes recruited to RNA pol II (polymerase II) during the early stages of transcription. We recently discovered that the mammalian cap methyltransferase is a heterodimer consisting of RNMT and the RNMT-activating subunit RAM (RNMT-activating mini-protein). RAM activates and stabilizes RNMT and thus is critical for cellular cap methylation and cell viability. In the present study we report that RNMT interacts with the N-terminal 45 amino acids of RAM, a domain necessary and sufficient for maximal RNMT activation. In contrast, smaller components of this RAM domain are sufficient to stabilize RNMT. RAM functions in the nucleus and we report that nuclear import of RAM is dependent on PY nuclear localization signals and Kapβ2 (karyopherin β2) nuclear transport protein.
Highlights
In eukaryotes, RNA pol polymerase II (II) transcripts are synthesized as precursors which undergo a complex series of processing events prior to translation
human embryonic kidney (HEK)-293 cells were transiently transfected with pcDNA4-based constructs using calcium phosphate. siRNA was purchased from the Dharmacon siGENOME collection [RNA guanine-7 methyltransferase (RNMT)-activating mini-protein (RAM), D-021286-01; RNMT, D-01952501; karyopherin β2 (Kapβ2)-1, D-011308-01; Kapβ2-2, D011308-02], and transfected using LipofectamineTM RNAiMAX (Invitrogen)
HeLa cells were depleted for endogenous RAM using RAM siRNA and transfected with pcDNA5-based constructs which express RAM–GFP or GFP alone (Figure 1B)
Summary
RNA pol II (polymerase II) transcripts are synthesized as precursors which undergo a complex series of processing events prior to translation. The first processing event is the addition of the cap, an inverted 7-methylguanosine group joined to the first transcribed nucleotide via a 5 to 5 triphosphate bridge [1,2,3,4]. In higher eukaryotes the first transcribed nucleotides are methylated in a variety of species-specific configurations. The cap is uniquely found on RNA pol II transcripts and is critical for transcript expression. The cap structure protects transcripts from exonucleases and recruits complexes, including CBC (cap-binding complex) and eIF4F (eukaryotic initiation factor 4F) complex, which mediate transcription elongation, splicing, nuclear export and translation initiation [1]. The cap is present on the transcript throughout its lifetime and the process of decapping initiates RNA degradation [5]
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