Abstract
THO/TREX connects transcription with genome integrity in yeast, but a role of mammalian THO in these processes is uncertain, which suggests a differential implication of mRNP biogenesis factors in genome integrity in yeast and humans. We show that human THO depletion impairs transcription elongation and mRNA export and increases instability associated with DNA breaks, leading to hyper-recombination and γH2AX and 53BP1 foci accumulation. This is accompanied by replication alteration as determined by DNA combing. Genome instability is R-loop–dependent, as deduced from the ability of the AID enzyme to increase DNA damage and of RNaseH to reduce it, or from the enhancement of R-loop–dependent class-switching caused by THOC1-depletion in CH12 murine cells. Therefore, mammalian THO prevents R-loop formation and has a role in genome dynamics and function consistent with an evolutionary conservation of the functional connection between these mRNP biogenesis factors and genome integrity that had not been anticipated.
Highlights
Transcription is a central cellular process occurring in the nucleus of eukaryotic cells in coordination with other nuclear processes
Our study reveals that depletion of human THO subunits, in particular THOC1/hHPR1, reduces transcription elongation, affects mRNA export, and increases genome instability associated with the accumulation of DNA breaks
After transfection THOC1, THOC5, UAP56 and ALY mRNA levels were reduced to 33%, 26%, 51% and 19%, respectively, compared to the levels of the cells transfected with the siC control (Figure 1A)
Summary
Transcription is a central cellular process occurring in the nucleus of eukaryotic cells in coordination with other nuclear processes. The nascent pre-mRNA associates with mRNA-binding proteins and undergoes a series of processing steps, resulting in export-competent mRNA ribonucleoprotein complexes (mRNP) that are transported into the cytoplasm. THO mutations lead to gene expression defects that are evident for long and GC-rich DNA sequences [3], as well as for repeat-containing genes [5] Such defects are the consequence of an impairment in transcription elongation as determined both in vivo and in vitro [3,6,7]. Yeast THO would participate in the co-transcriptional formation of export-competent mRNP during transcription elongation by controlling the assembly of heterogeneous nuclear ribonucleoproteins (hnRNPs) onto the mRNA [10]
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