DISCOVERY OF A NOVEL WAY TO CONTROL GENES IN CELL - "BACKTRACKING"

 

Humans have 30 trillion cells each containing a set of tiny molecules made of sugar and a base as a backbone known as DNA. However not all cells function the same or look the same due to the regulation of gene expression - gene being expressed when needed and vice-versa. So for every cell, a particular set of genes is only expressed for their function in the body, this activity of controlling gene expression is been regulated by many factors. However, scientists have discovered an unknown way by which this regulation is accomplished.

Humans with 20,000 to 25,000 genes have varied sizes ranging from a few hundred bases to 2 million bases, to function appropriately they need to be copied down, and transcribed into molecules called RNA. These RNAs get translated into protein which is the primary requirement for a cell to function. To do all these functions, certain enzymes are needed. One such enzyme is RNA Polymerase II (RNA Pol II) which translates DNA into RNA of protein-coding genes or noncoding RNAs essential in the case of eukaryotes. 

RNA Pol II sometimes face obstacles in translation due to DNA damage, secondary structure in DNA, or binding of proteins in the DNA which contribute to pause or arrest. On this condition, the enzyme backtracks along the template by which the 3’ end of the RNA is been exposed from the catalytic site of the RNA Pol II. However, this elongation can be restored by transcription factor IIS (TFIIS) which promotes the cleavage of the exposed backtracked nascent RNA, thereby creating the 3’ end in the catalytic site of RNA Poll II. 

Backtracking mechanism

 

Backtracking is found both in eukaryotes and bacteria and has been associated with DNA repair and gene regulation. Moreover, this exposed 3’ RNA due to backtracking can disrupt the transcription which may delay the production of cellular protein, breaking the balance. In the case of bacteria and yeast, transcription and replication machinery run alongside, so backtracking can lead to a collision of the machinery leading to DNA break and genomic instability.  

In-vitro studies have stated that RNA Pol II possesses intrinsic cleavage activity which helps to resolve the backtracking on its own and on longer backtracking, the TFIIS factor helps in eradicating the nascent transcript. It has also been identified that backtracking often occurs in bacteria and yeast too. 

Through previous studies, it has been found that a longer shift of the 3’ end takes more time to process and resolve and cleavage of the 3’ exposed end is difficult due to the presence of RNA Pol II. Characterisation of backtracking has been previously studied, however, the mechanism behind the backtracking is been discovered but not backtracking itself.  Especially the position where the RNA Pol II is positioned when backtracking is still unknown. So the mechanism for resolving backtracking is unclear and the prominent occurrence of it is also unknown till now. 

This uncertainty on backtracking is over as a paper has been published in Molecular Cell titled “Persistence of backtracking by human RNA polymerase II” where they have mapped and characterised the persistent backtracking in mammalian cells using long-range cleavage sequencing (LORAX-seq), a method by which direct backtracked RNA can be sequenced from beginning to end in in-vitro. 

LORAX-seq Method

Through their data, they have stated that persistent backtracking occurs when RNA Pol II pauses but pause strength doesn't determine the potential of the backtracking. They have also shown that the backtracking prominently occurs near promoters and intron-exon junctions. Moreover, researchers found that persistent backtracking during transcription isn't random. It tends to happen more often in specific genes linked to tasks like making proteins, regulating the cell cycle, building nucleosomes (the units that package DNA), and controlling development. When persistent backtracking isn't fixed in cells, the genes related to these tasks don't work as well. Particularly, genes responsible for making histones (proteins crucial for DNA packaging) are very prone to persistent backtracking. The study showed that managing the buildup and resolution of these backtracking events is crucial for ensuring histones are made at the right time during DNA replication. Additionally, through LORAX-seq they have identified that RNA Pol II slides backwards on pause with a 20 nucleotide distance from the 3’ end. 

In conclusion, the application of LORAX-seq in various contexts, such as ageing or disease, has provided valuable insights into the impact of persistent backtracking on biological processes. Through this technique, researchers have elucidated how persistent backtracking affects ageing-related phenomena like the decline in the heat shock response and defective histone biosynthesis. Moreover, unresolved persistent backtracking in ageing and cancer has been linked to genome instability, chromatin remodelling, and gene dysregulation. These findings underscore the importance of further exploration into the role of persistent backtracking, as it offers a deeper understanding of its implications in ageing, disease progression, and cellular function.

REFERENCE

Yang KB, Rasouly A, Epshtein V, Martinez C, Nguyen T, Shamovsky I, et al. Persistence of backtracking by human RNA polymerase II. Molecular Cell. 2024 Mar;84(5). doi:10.1016/j.molcel.2024.01.019 

IMAGE CREDIT

Stanford Medicine - Stanford University, https://images.app.goo.gl/6QJaiF6JSoRu1mdC9

Semantic Scholar, https://images.app.goo.gl/V2W8We8bgJqg6zEf8

Molecular Cell, https://images.app.goo.gl/TRSRWeS9NfMVGAsZ8