Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA.
Cells are continuously exposed to both endogenous and exogenous sources of genomic stress. To maintain chromosome stability, a variety of mechanisms have evolved to cope with the multitude of genetic abnormalities that can arise over the life of a cell. Still, failures to repair these lesions are the driving force of cancers and other degenerative disorders. DNA double-strand breaks (DSBs) are among the most toxic genetic lesions, inhibiting cell ability to replicate, and are sites of mutations and chromosomal rearrangements. DSB repair is known to proceed via two major mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ). HR reliance on the exchange of genetic information between two identical or nearly identical DNA molecules offers increased accuracy. While the preferred substrate for HR in mitotic cells is the sister chromatid, this is limited to the S and G2 phases of the cell cycle. However, abundant amounts of homologous genetic substrate may exist throughout the cell cycle in the form of RNA. Considered an uncommon occurrence, the direct transfer of information from RNA to DNA is thought to be limited to special circumstances. Studies have shown that RNA molecules reverse transcribed into cDNA can be incorporated into DNA at DSB sites via a non-templated mechanism by NHEJ or a templated mechanism by HR. In addition, synthetic RNA molecules can directly template the repair of DSBs in yeast and human cells via an HR mechanism. New work suggests that even endogenous transcript RNA can serve as a homologous template to repair a DSB in chromosomal DNA. In this perspective, we will review and discuss the recent advancements in DSB repair by RNA via non-templated and templated mechanisms. We will provide current findings, models and future challenges investigating RNA and its role in DSB repair.
The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells transcribe abundant amount of RNA with a wide range of functions, it has been difficult to uncover whether RNA can serve as a template for DNA repair and recombination. An increasing number of experimental evidences suggest a direct role of RNA in DNA modification. Recently, we demonstrated that endogenous transcript RNA can serve as a template to repair a DNA double-strand break (DSB), the most harmful DNA lesion, not only indirectly via formation of a DNA copy (cDNA) intermediate, but also directly in a homology driven mechanism in budding yeast. These results point out that the transfer of genetic information from RNA to DNA is more general than previously thought. We found that transcript RNA is more efficient in repairing a DSB in its own DNA (in cis) than in a homologous but ectopic locus (in trans). Here, we summarize current knowledge about the process of RNA-driven DNA repair and recombination, and provide further data in support of our model of DSB repair by transcript RNA in cis. We show that a DSB is precisely repaired predominately by transcript RNA and not by residual cDNA in conditions in which formation of cDNA by reverse transcription is inhibited. Additionally, we demonstrate that defects in ribonuclease (RNase) H stimulate precise DSB repair by homologous RNA or cDNA sequence, and not by homologous DNA sequence carried on a plasmid. These results highlight an antagonistic role of RNase H in RNA-DNA recombination. Ultimately, we discuss several questions that should be addressed to better understand mechanisms and implications of RNA-templated DNA repair and recombination.
Highlights d DNA polymerase z promotes RNA-templated DNA repair and modification d cDNA-, unlike RNA-templated DNA repair, requires endclipping function d RNA-mediated DNA modification proceeds in the absence of recombination genes d Mismatch repair facilitates accurate repair with template RNA
Insertion sequences (IS) are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance. IS200/IS605 elements undergo peel-and-paste transposition catalyzed by a TnpA transposase, but intriguingly, they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively. Recent studies demonstrated that TnpB-family enzymes function as RNA-guided DNA endonucleases, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB/IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related IS elements from Geobacillus stearothermophilus that encode diverse TnpB/IscB orthologs, and showed that a single TnpA transposase was active for transposon excision. The donor joints formed upon religation of IS-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB/IscB nucleases, and co-expression of TnpB together with TnpA led to significantly greater transposon retention, relative to conditions in which TnpA was expressed alone. Remarkably, TnpA and TnpB/IscB recognize the same AT-rich transposon-adjacent motif (TAM) during transposon excision and RNA-guided DNA cleavage, respectively, revealing a striking convergence in the evolution of DNA sequence specificity between collaborating transposase and nuclease proteins. Collectively, our study reveals that RNA-guided DNA cleavage is a primal biochemical activity that arose to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defense.
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