G-quadruplex structures in RNA stimulate mitochondrial transcription termination and primer formation

Abstract

The human mitochondrial transcription machinery generates the primers required for initiation of leading-strand DNA replication. According to one model, the 3′ end of the primer is defined by transcription termination at conserved sequence block II (CSB II) in the mitochondrial DNA control region. We here demonstrate that this site-specific termination event is caused by G-quadruplex structures formed in nascent RNA upon transcription of CSB II. We also demonstrate that a poly-dT stretch downstream of CSB II has a modest stimulatory effect on the termination efficiency. The mechanism is reminiscent of Rho-independent transcription termination in prokaryotes, with the exception that a G-quadruplex structure replaces the hairpin loop formed in bacterial mRNA during transcription of terminator sequences. mitochondrion | RNA polymerase H uman mitochondria contain their own genetic material called mitochondrial DNA (mtDNA), a double-stranded circular molecule encoding 13 subunits of the respiratory chain, 22 tRNAs, and 2 rRNAs. The two strands are referred to as the heavy and light strand based on their buoyant density in cesium chloride gradients (1). Transcription from the light strand promoter (LSP) and heavy strand promoter is polycistronic and produces near genome-length transcripts, which are processed to release the individual RNA molecules (2, 3). MtDNA contains few noncoding sequences, with the exception of the control region, which harbors DNA elements required for initiation of transcription and DNA replication (Fig. 1A).Mitochondrial DNA replication initiates at two major sites, the origins of light and heavy (OriH) strand replication. The mitochondrial RNA polymerase generates the RNA primers required for initiation of DNA synthesis at both these sites (4-7). Primers required for OriH activation are produced by transcription initiated at LSP, and there must thus exist a mechanism that enables the transcription machinery to switch from genomelength transcription to primer formation. One model suggests that the primers are formed by cleavage of the primary LSP transcript by the mitochondrial RNA processing endonuclease (8, 9), but this idea has been questioned, because at least human mitochondrial RNA processing endonuclease is primarily localized to the nucleolus, where it is required for rRNA processing (10). Some years ago, we suggested an alternative model for primer formation at OriH. Using a reconstituted in vitro transcription system containing the mitochondrial RNA polymerase (POLRMT) and mitochondrial transcription factors A (TFAM) and B2 (TFB2M), we observed that a large fraction (up to 65%) of the transcription events initiated at LSP were prematurely terminated at a conserved DNA element within the control region, denoted conserved sequence block II (CSB II). The 3′ ends of these terminated transcription products coincided with the RNA-to-DNA transition sites mapped in vivo (5). The mechanism behind the observed transcription termination was not elucidated, but mutational analysis demonstrated ...

DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase

“…Mitochondrial transcription creates primers in a CG-rich element, generally termed conserved sequence block II (CSBII), required for the initiation of leading strand DNA replication. G-quadruplexes formed by RNA result in transcription termination and dictate primer formation for DNA synthesis initiation (77). More recently, Wanrooij et al (78) reported that the mitochondrial RNA primer required for the leading strand origin of mtDNA replication forms a hybrid G-quadruplex between RNA and DNA in CSBII and that this quadruplex formation plays a critical role in determining the rate of DNA synthesis initiation in human mitochondria by influencing the architecture and persistence of an RNA-DNA hybrid (R-loop) residing at the leading strand origin of DNA replication.…”

Genetic aspects of mitochondrial genome evolution

“…The mitochondrial transcription machinery, finally, also generates the RNA primer needed for initiation of heavy strand DNA synthesis. To this end, in mammals, a G-quadruplex structure is utilised to prematurely terminate transcription from the L-strand promoter (Wanrooij et al, 2010).…”

An RNA G-quadruplex in the 3' UTR of the proto-oncogene PIM1 represses translation

“…[1][2][3][4][5][6][7][8][9][10][11] In addition, RNA G-quadruplex structures can also stimulate mitochondrial transcription termination 12 and regulate alternative splicing in TP53 intron 3. 13 In addition, a G-quadruplex close to a cleavage sites in the 3' UTR of insulinlike growth factor II mRNA has been proposed and its structure formation has been demonstrated in vitro.…”