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The emerging role of RNAs in DNA damage repair

View Article: PubMed Central - PubMed

ABSTRACT

Many surveillance and repair mechanisms exist to maintain the integrity of our genome. All of the pathways described to date are controlled exclusively by proteins, which through their enzymatic activities identify breaks, propagate the damage signal, recruit further protein factors and ultimately resolve the break with little to no loss of genetic information. RNA is known to have an integral role in many cellular pathways, but, until very recently, was not considered to take part in the DNA repair process. Several reports demonstrated a conserved critical role for RNA-processing enzymes and RNA molecules in DNA repair, but the biogenesis of these damage-related RNAs and their mechanisms of action remain unknown. We will explore how these new findings challenge the idea of proteins being the sole participants in the response to DNA damage and reveal a new and exciting aspect of both DNA repair and RNA biology.

No MeSH data available.


Schematics of reporter systems used for small RNA sequencing experiments. (a) Wei et al.9 DR-GFP genomically integrated reporter. Transfection of I-SceI results in cleavage near the transcriptional start site (TSS) of GFP. Small RNA was sequenced and mapped back to the reporter sequence. (b) Francia et al.11 genomically integrated Lac-/Tet-operator-flanked I-SceI site. This reporter lacks transcriptional activity but is highly repetitive. Small RNA was detected after I-SceI transfection but at low levels (47 total reads after transfection versus 20 reads without). No information on where these RNAs mapped to was provided. (c) Michalik et al.13Drosophila expression plasmids, either circularised or linearised. Here only the BamHI linearised vector is shown as it produced the highest number of small RNA reads. Grey dashed lines represent the approximate distribution of small RNA mapping back to the locus, where positional data was supplied in the manuscript. Right-angled arrows represent TSS, whereas vertical wavy line denotes integration within the genome
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fig3: Schematics of reporter systems used for small RNA sequencing experiments. (a) Wei et al.9 DR-GFP genomically integrated reporter. Transfection of I-SceI results in cleavage near the transcriptional start site (TSS) of GFP. Small RNA was sequenced and mapped back to the reporter sequence. (b) Francia et al.11 genomically integrated Lac-/Tet-operator-flanked I-SceI site. This reporter lacks transcriptional activity but is highly repetitive. Small RNA was detected after I-SceI transfection but at low levels (47 total reads after transfection versus 20 reads without). No information on where these RNAs mapped to was provided. (c) Michalik et al.13Drosophila expression plasmids, either circularised or linearised. Here only the BamHI linearised vector is shown as it produced the highest number of small RNA reads. Grey dashed lines represent the approximate distribution of small RNA mapping back to the locus, where positional data was supplied in the manuscript. Right-angled arrows represent TSS, whereas vertical wavy line denotes integration within the genome

Mentions: How can the generation of small RNA in a DNA damage-specific context be detected? Commonly used external DNA damage agents, such as IR, lead to the generation of multiple breaks at random genomic sites. This makes the task of discovery of novel RNA species with the use of an NGS approach virtually impossible, as breaks need to occur in known defined sequences for this experimental strategy to work. To date, three studies have used restriction enzyme-based systems in animal cells combined with NGS to detect diRNAs, which map to the vicinity of the cut site.9, 11, 13 These reports relied on the ectopic expression of rare restriction enzymes targeted to specific pre-integrated loci in the genome. Two different systems were adopted in human cell lines: the DR-GFP HR reporter or a Lac-/Tet-operator repeat-flanking reporter,22, 23 both of which contain a single recognition site for the uniquely cutting meganuclease I-SceI (Figures 3a and b, see also Figure 4 and the more in-depth discussion of the DR-GFP reporter assay below). Following the transfection and expression of I-SceI for 12 to 24 h, small RNAs were sequenced by NGS. These two studies reported the existence of small RNAs around the break sites.9, 11 However, the exact roles of these small RNAs are yet to be determined.


The emerging role of RNAs in DNA damage repair
Schematics of reporter systems used for small RNA sequencing experiments. (a) Wei et al.9 DR-GFP genomically integrated reporter. Transfection of I-SceI results in cleavage near the transcriptional start site (TSS) of GFP. Small RNA was sequenced and mapped back to the reporter sequence. (b) Francia et al.11 genomically integrated Lac-/Tet-operator-flanked I-SceI site. This reporter lacks transcriptional activity but is highly repetitive. Small RNA was detected after I-SceI transfection but at low levels (47 total reads after transfection versus 20 reads without). No information on where these RNAs mapped to was provided. (c) Michalik et al.13Drosophila expression plasmids, either circularised or linearised. Here only the BamHI linearised vector is shown as it produced the highest number of small RNA reads. Grey dashed lines represent the approximate distribution of small RNA mapping back to the locus, where positional data was supplied in the manuscript. Right-angled arrows represent TSS, whereas vertical wavy line denotes integration within the genome
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384027&req=5

fig3: Schematics of reporter systems used for small RNA sequencing experiments. (a) Wei et al.9 DR-GFP genomically integrated reporter. Transfection of I-SceI results in cleavage near the transcriptional start site (TSS) of GFP. Small RNA was sequenced and mapped back to the reporter sequence. (b) Francia et al.11 genomically integrated Lac-/Tet-operator-flanked I-SceI site. This reporter lacks transcriptional activity but is highly repetitive. Small RNA was detected after I-SceI transfection but at low levels (47 total reads after transfection versus 20 reads without). No information on where these RNAs mapped to was provided. (c) Michalik et al.13Drosophila expression plasmids, either circularised or linearised. Here only the BamHI linearised vector is shown as it produced the highest number of small RNA reads. Grey dashed lines represent the approximate distribution of small RNA mapping back to the locus, where positional data was supplied in the manuscript. Right-angled arrows represent TSS, whereas vertical wavy line denotes integration within the genome
Mentions: How can the generation of small RNA in a DNA damage-specific context be detected? Commonly used external DNA damage agents, such as IR, lead to the generation of multiple breaks at random genomic sites. This makes the task of discovery of novel RNA species with the use of an NGS approach virtually impossible, as breaks need to occur in known defined sequences for this experimental strategy to work. To date, three studies have used restriction enzyme-based systems in animal cells combined with NGS to detect diRNAs, which map to the vicinity of the cut site.9, 11, 13 These reports relied on the ectopic expression of rare restriction enzymes targeted to specific pre-integrated loci in the genome. Two different systems were adopted in human cell lines: the DR-GFP HR reporter or a Lac-/Tet-operator repeat-flanking reporter,22, 23 both of which contain a single recognition site for the uniquely cutting meganuclease I-SceI (Figures 3a and b, see also Figure 4 and the more in-depth discussion of the DR-GFP reporter assay below). Following the transfection and expression of I-SceI for 12 to 24 h, small RNAs were sequenced by NGS. These two studies reported the existence of small RNAs around the break sites.9, 11 However, the exact roles of these small RNAs are yet to be determined.

View Article: PubMed Central - PubMed

ABSTRACT

Many surveillance and repair mechanisms exist to maintain the integrity of our genome. All of the pathways described to date are controlled exclusively by proteins, which through their enzymatic activities identify breaks, propagate the damage signal, recruit further protein factors and ultimately resolve the break with little to no loss of genetic information. RNA is known to have an integral role in many cellular pathways, but, until very recently, was not considered to take part in the DNA repair process. Several reports demonstrated a conserved critical role for RNA-processing enzymes and RNA molecules in DNA repair, but the biogenesis of these damage-related RNAs and their mechanisms of action remain unknown. We will explore how these new findings challenge the idea of proteins being the sole participants in the response to DNA damage and reveal a new and exciting aspect of both DNA repair and RNA biology.

No MeSH data available.