Transcription termination:

Forgotten mechanism in RNA synthesis cycle 

 

Takayuki Nojima, Associate Professor

Cancer Genome Regulation, MiB

 

Abstract

Transcription is terminated in appropriate region to prevent genome stresses such as transcription-replication conflict and RNA-DNA hybrid caused in extragenic region of eukaryote cells (Ref 1, 2). Biochemical and genetic approaches identified several trans-factors and cis-elements involved in transcription termination. However, the mechanism and the rule remain largely unclear due to technical limitation and complexity created by other co-transcriptional events.

 

In order to investigate precise mechanisms of transcription, I developed a nascent RNA technology named mammalian native elongating transcript-sequencing (mNET-seq) method that reveals Pol II pausing and RNA processing intermediates with the phosphorylation states at single nucleotide resolution (Ref 3). In mNET-seq analysis, RNA polymerase II (Pol II) machinery was specifically detected at termination region of protein coding genes with the CTD phosphorylation at threonine 4 position (T4P). On the other hand, the T4P CTD mark is distributed throughout long noncoding RNA (lncRNA) gene units, suggesting transcription termination signals are embedded in lncRNA genes to fine tune lncRNA expression (Ref 4).

 

I substantially extended mNET-seq method to dissect intact nascent RNA associated with elongating Pol II machinery. This new method was termed Polymerase Intact Nascent Transcript (POINT) technology (Ref 5). The POINT technology is applied to a template switching based 5’RACE method, resulting in detection of nascent transcript 5’ends at single nucleotide resolution (POINT-5) in illumina platform. Therefore POINT-5 method precisely profiles transcription start sites and co-transcriptional RNA cleavage sites. Notably rapid depletion of nuclear 5’-3’ exonuclease Xrn2 significantly induced a termination defect with increased RNA cleavage peaks only on polyadenylation sites of pre-mRNA genes, but not on other cleavage sites. This analysis revealed specificity of Xrn2-dependent RNA turnover. 

 

Finally, I here will share some preliminary results of transcription termination regulation in specialized chromatin environments.

 

 

References (* corresponding authors)

1. Nojima T* and Proudfoot NJ*.

Mechanism of lncRNA biogenesis as revealed by nascent transcriptomics. Nature Reviews Molecular Cell Biology, 23, 389-406, 2022

2. Nojima T*, Tellier M, Foxwell J, Rebeiro deA C, Tan-Wong SM, Dhir S, Dhir A, Murphy S* and Proudfoot NJ*.

Deregulated expression of lncRNA through loss of SPT6 induces R-loop, DNA replication stress and cellular senescence. Molecular Cell, 72:1-15, 2018

3. Nojima T, Gomes T, Fialho Grosso AR, Kimura H, Dye MJ, Dhir S, Carmo-Fonseca M* and Proudfoot NJ*. Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing. Cell, 23; 526-40, 2015
4. Schlackow M, Nojima T*, Gomes T, Dhir A, Carmo-Fonseca and Proudfoot NJ*.

Distinctive patterns of transcription and RNA processing for human lincRNA, Molecular Cell, 65:25-38, 2017

5. Sousa-Luis R, Dujardin G, Zukher I, Kimura H, Carmo-Fonseca M*, Proudfoot NJ*, and Nojima T*.

POINT Technology Illuminates the Processing of Polymerase-Associated Intact Nascent Transcripts. Molecular Cell, 81, 1935-1950, 2021