Related Experiment Video
Updated: Jun 2, 2026
10:59
Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events
Published on: May 13, 2019
A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors
Cristina Tous1, Ana G Rondón, María García-Rubio
1Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain.
The EMBO Journal
|April 12, 2011
Summary
This study introduces a new G-less-based run-on (GLRO) assay to analyze transcription elongation in vivo. The assay reveals that mRNA processing factors and the Nup84 complex are crucial for efficient elongation, linking RNA processing and nuclear export to transcription.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
- Chromatin Dynamics
Background:
- Transcription elongation is a critical step in gene expression, influenced by various protein factors.
- Understanding the interplay between mRNA processing, nuclear export, and transcription elongation is essential for comprehending gene regulation.
Purpose of the Study:
- To develop and validate a novel in vivo assay for direct analysis of transcription elongation on chromatin.
- To investigate the roles of specific mRNA processing factors and the Nup84 complex in transcription elongation.
Main Methods:
- Development of the G-less-based run-on (GLRO) assay using specific DNA substrates.
- Application of the GLRO assay to analyze the requirement of various factors in transcription elongation.
- Confirmation using RNA polymerase II chromatin immunoprecipitations and mutant screening.
Main Results:
- The GLRO assay proved to be a sensitive and reliable method for in vivo elongation analysis.
- Identified PAF, THSC/TREX-2, SAGA, Rrp6, Rna14, and Rna15 as factors required for efficient transcription elongation.
- Discovered a novel requirement for the Nup84 nucleoporin complex in transcription elongation.
Conclusions:
- The study validates the GLRO assay for studying transcription elongation.
- Provides new evidence for the involvement of specific mRNA processing factors in transcription elongation.
- Establishes a new role for the Nup84 complex, supporting a link between pre-mRNA processing, nuclear export, and transcription elongation.
Related Concept Videos
02:35Transcription Elongation Factors
Transcription elongation is a dynamic process that alters depending upon the sequence heterogeneity of the DNA being transcribed. Hence, it is not surprising that the elongation complex's composition also varies along the way while transcribing a gene.
The transcription elongation is regulated via pausing of RNA polymerase on several occasions during transcription. In bacteria, these halts are necessary because the transcription of DNA into mRNA is coupled to the translation of that mRNA into a...
The transcription elongation is regulated via pausing of RNA polymerase on several occasions during transcription. In bacteria, these halts are necessary because the transcription of DNA into mRNA is coupled to the translation of that mRNA into a...
02:35Transcription Elongation Factors
Transcription elongation is a dynamic process that alters depending upon the sequence heterogeneity of the DNA being transcribed. Hence, it is not surprising that the elongation complex's composition also varies along the way while transcribing a gene.
The transcription elongation is regulated via pausing of RNA polymerase on several occasions during transcription. In bacteria, these halts are necessary because the transcription of DNA into mRNA is coupled to the translation of that mRNA into a...
The transcription elongation is regulated via pausing of RNA polymerase on several occasions during transcription. In bacteria, these halts are necessary because the transcription of DNA into mRNA is coupled to the translation of that mRNA into a...
02:27Nonsense-mediated mRNA Decay
The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
02:31Nuclear Export of mRNA
Before mRNAs are exported to the cytoplasm, it is crucial to check each mRNA for structural and functional integrity. Eukaryotic cells use several different mechanisms, collectively known as mRNA surveillance, to look for irregularities in mRNAs. Irregular or aberrant mRNA are rapidly degraded by various enzymes. If a defective mRNA escapes the surveillance, it would be translated into a protein which would either be non-functional or not function properly. One of the primary irregularities in...
02:41Chromatin Structure Regulates pre-mRNA Processing
In eukaryotic cells, nascent mRNA transcripts need to undergo many post-transcriptional modifications to reach the cell cytoplasm and translate into functional proteins. For a long time, transcription and pre-mRNA processing were considered two independent events that occur sequentially in the cell. However, it has now been well established that transcription and pre-mRNA processing are two simultaneous processes that are precisely regulated inside the cell.
The chromatin structure, especially...
The chromatin structure, especially...
02:53Ribosomal RNA Synthesis
Ribosome synthesis is a highly complex and coordinated process involving more than 200 assembly factors. The synthesis and processing of ribosomal components occurs not only in the nucleolus but also in the nucleoplasm and the cytoplasm of eukaryotic cells.
Ribosome biogenesis begins with the synthesis of 5S and 45S pre-rRNAs by distinct RNA polymerases. The primary transcripts are extensively processed and modified before they are bound and folded by ribosomal proteins and assembly factors,...
Ribosome biogenesis begins with the synthesis of 5S and 45S pre-rRNAs by distinct RNA polymerases. The primary transcripts are extensively processed and modified before they are bound and folded by ribosomal proteins and assembly factors,...