The researchers say they have developed a new technique that, they claim, can determine for the first time how often and exactly where molecular events called "backtracking" occur in the genome of any species. Their findings, "Retrospective persistence of human RNA polymerase II," appeared in the journal Molecular Cell, supporting the theory that backtracking represents a broad form of gene regulation that affects thousands of human genes, including many involved in fundamental life processes, such as cell** and uterine development, according to a team led by scientists at NYU Grossman School of Medicine.
In 1997, Dr. Evgeny Nudler and colleagues published a paper showing that RNA polymerase sometimes slides backwards along the strand it is reading, which they call "backtracking." Studies since then have shown that backtracking occasionally occurs in living cells shortly after the RNA polymerase begins RNA synthesis, or when it encounters damaged DNA to make room for afferent repair enzymes. Subsequent studies have shown that the regression and repair mechanism must work quickly and dissipate otherwise it may collide with DNA polymerases, resulting in cell death-induced breaks in the DNA strands.
Now, this new study, led by the Nudler team at NYU Langone Health, shows that their new technology, long-range cut sequencing (LoRax-Seq), can directly detect where a backtracking event begins and ends. By complementing past indirect or limited methods, the new approach reveals many such events farther than previously thought, and that doing so lasts much longer. The findings also suggest that sustained backtracking occurs frequently throughout the genome, more frequently near certain gene types, and that its function extends far beyond DNA repair.
RNA polymerase II (RNA POL II) can be traced back during transcriptional elongation, exposing nascent RNA3'Extremity. Nascent RNA sequencing can approximate the location of fast-resolving backtracking events; However, the extent of more persistent backtracking and genome-wide distribution are unknown. Therefore, we have developed a direct response to extrusion'Retrospective'3 methods of sequencing RNA," the researchers wrote.
Our data suggest that RNA POL II slides backward for more than 20 nt in human cells and can persist in this regressive state. Sustained backtracking occurs primarily when RNA POL II stalls near promoter and intron-exon junctions and is enriched in genes involved in translation, replication, and development, and if these events are not resolved, gene expression is reduced. Histone genes are highly susceptible to ongoing backtracking, and timely expression during cellular processes may require resolution of such events.
These results suggest that continuous backtracking may affect different gene expression programs.
The astonishing stability of backtracking over greater distances makes it possible that it represents a ubiquitous form of genetic regulation in species ranging from bacteria to humans," said Nudler, senior author of the study and Professor Julie Wilson Anderson in the Department of Biochemistry and Molecular Pharmacology at NYU Langone. "If further work extends our findings to different developmental programs and pathological conditions, backtracking could resemble epigenetics, whose findings reveal a surprising new layer of gene regulation without altering the DNA code.
Past studies have shown that when RNA polymerase II is backtracked, it extrudes the tip of its DNA-based RNA strand from its internal channels. Since prolonged backtracking is prone to harmful collisions, transcription is thought to be rapidly restored by transcription factor IIS (TFIIS), which facilitates the cleavage (cleavage) of the extruded "traceback" RNA. This clears the way for RNA polymerase II to resume its forward** reads.
However, other early studies have shown that when the polymerase backtracks beyond a certain distance (e.g., 20 nucleobase DNA building blocks), the retraced RNA can attach to the channel from which it was squeezed out, allowing it to remain held longer. Locked, retraced complexes are less likely to be rescued by TFIS-driven cleavage and more likely to delay transcription of related genes.
This has led to the theory that backtracking, in addition to playing a key role in the DNA repair pathway, can also act as a major regulatory mechanism to up- or down-modulate the role of genes.
According to the researchers, TFIIS may occur in low concentrations in living cells and compete with hundreds of other proteins to obtain and cut off the backtracked RNA so that transcription can continue. In the current study, the team used a high concentration of purified TFIIS (non-competing protein) to precisely cleave any retraced RN** segment so that it appears anywhere in the cell's genetic code. This makes the cut fragment a technique that can be used to read the sequence and provide clues to its position and function.
The team also found that genes that control histones are highly susceptible to continuous backtracking. The authors speculate that the extent to which this occurs, as well as the associated changes in the transcription of certain genes, may control the timing of the massive histone accumulation required by cells to rebuild chromatin. They also showed that continuous backtracking may affect the timely transcription of genes critical for tissue development.
In addition to its potentially useful functions, sustained backtracking can lead to DNA damage and other disease-leading genetic dysfunctions," said first study author Kevin Yang, a graduate student in Nudler's lab. "We speculate, for example, that measuring traceback in the context of aging or cancer may help us understand why failures occur in cellular stress responses and cell replication, and propose new methods.
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