MRNA Cap Methyltransferase, RNMT-RAM, Promotes RNA Pol II-Dependent Transcription

Dhaval Varshney,Olivia Lombardi,Gabriele Schweikert,Sianadh Dunn,Olga Suska,Victoria H Cowling

doi:10.1016/j.celrep.2018.04.004

Dhaval Varshney, Olivia Lombardi + Show 4 more

Open Access

https://doi.org/10.1016/j.celrep.2018.04.004

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Journal: Cell Reports	Publication Date: May 1, 2018
Citations: 37	License type: cc-by

Affiliation: University of Dundee, University of Edinburgh

Abstract

SummarymRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription.

Highlights

During the initial stages of eukaryotic pre-mRNA transcription, nascent transcripts are modified by mRNA cap addition (Furuichi, 2015; Shuman, 2015)
RNMT-RAM Promotes RNA Pol II-Dependent Transcription To investigate the cellular role of the mRNA cap methyltransferase, RNMT-RAM, HeLa cells were transfected with two RAM small interfering RNAs, which reduce expression of RNMT and RAM (Figure 1A) (Gonatopoulos-Pournatzis et al, 2011)
While RAM was depleted by RAM siRNA transfection, HA-tagged RNMT (HA-RNMT) was induced from a doxycycline-regulated promoter (Figure 1B)

Summary

Introduction

During the initial stages of eukaryotic pre-mRNA transcription, nascent transcripts are modified by mRNA cap addition (Furuichi, 2015; Shuman, 2015). The mRNA cap protects transcripts from 50 exonucleases and recruits factors involved in splicing, nuclear export, and translation initiation (Gonatopoulos-Pournatzis and Cowling, 2014a; Topisirovic et al, 2011; Brannan et al, 2012). The S. cerevisiae cap methyltransferase, ABD1, is important for transcription of certain genes (Schroeder et al, 2004). A mechanism has not been defined, the transcriptional defects resulting from ABD1 inactivation are rescued by the methyltransferase-dead enzyme, demonstrating independence from mRNA cap methylation. The S. pombe cap methyltransferase, PCM1, stimulates transcription by recruiting the elongation factor P-TEFb (Guiguen et al, 2007). A role for the mammalian mRNA cap methyltransferase in transcription has not been observed, it is recruited to transcription initiation sites (Aregger and Cowling, 2013; Glover-Cutter et al, 2008)

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