Human THO coordinates transcription termination and subsequent transcript release from the HSP70 locus.

Area of Science:

• Molecular Biology
• Gene Expression
• RNA Processing

Background:

• The THO/TREX complex plays a vital role in coupling transcription, 3'-end formation, and mRNA nuclear export.
• Understanding the precise mechanisms by which THO/TREX regulates these processes is essential for comprehending gene expression control.

Purpose of the Study:

• To investigate the role of THOC5, a component of the human THO complex (hTHO), in mRNA processing and transcriptional termination.
• To identify other key factors that interact with hTHO and contribute to mRNA release.

Main Methods:

• Copurification of mRNA processing factors with hTHO.
• Chromatin immunoprecipitation (ChIP) to assess protein-crosslinking at specific gene loci.
• siRNA-mediated knockdown to deplete specific proteins (THOC5, XRN2, DDX5/DDX17).
• Analysis of HSP70 transcript levels and localization.

Main Results:

• XRN2, DDX5/DDX17, and CstF64 were found to copurify with hTHO.
• Down-regulation of THOC5 led to increased cross-linking of XRN2 and CstF64 at the HSPA1A gene's RNAP II pause site.
• Knockdown of XRN2 or DDX5/DDX17 impaired HSP70 transcript release and promoted read-through transcription.
• Overexpression of RNase H reduced nuclear accumulation of HSP70 transcripts in depleted cells.

Conclusions:

• hTHO, in conjunction with DDX5/DDX17, plays a role in restricting R-loop formation.
• This action facilitates XRN2-mediated transcriptional termination and the release of mature transcripts from the HSPA1A gene.
• The THO/TREX complex is a key regulator of efficient and accurate gene expression through its influence on transcription termination.