THO and TRAMP complexes prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions

Expansion of structure-forming CAG/CTG repetitive sequences is the cause of several neurodegenerative disorders and deletion of repeats is a potential therapeutic strategy. Transcription-associated mechanisms are known to cause CAG repeat instability. In this study, we discovered that Thp2, an RNA export factor and member of the THO complex, and Trf4, a key component of the TRAMP complex involved in nuclear RNA degradation, are necessary to prevent CAG fragility and repeat contractions in a S. cerevisiae model system. Depletion of both Thp2 and Trf4 proteins causes a highly synergistic increase in CAG repeat fragility, indicating a complementary role of the THO and TRAMP complexes in preventing genome instability. Loss of either Thp2 or Trf4 causes an increase in RNA polymerase stalling at the CAG repeats and genome-wide transcription-replication conflicts (TRCs), implicating impairment of transcription elongation as a cause of CAG fragility and instability in their absence. Analysis of the effect of RNase H1 overexpression on CAG fragility and TRCs suggests that co-transcriptional R-loops are the main cause of CAG fragility in the thp2Δ mutants. In contrast, CAG fragility and TRCs in the trf4Δ mutant can be compensated for by RPA overexpression, suggesting that excess unprocessed RNA in TRAMP4 mutants leads to reduced RPA availability and high levels of TRCs. Our results show the importance of RNA surveillance pathways in preventing RNAPII stalling, TRCs, and DNA breaks, and show that RNA export and RNA decay factors work collaboratively to maintain genome stability.

Substrate discrimination and quality control require each catalytic activity of TRAMP and the nuclear RNA exosome

Quality control requires discrimination between functional and aberrant species to selectively target aberrant substrates for destruction. Nuclear RNA quality control in Saccharomyces cerevisiae includes the TRAMP complex that marks RNA for decay via polyadenylation followed by helicase-dependent 3′ to 5′ degradation by the RNA exosome. Using reconstitution biochemistry, we show that polyadenylation and helicase activities of TRAMP cooperate with processive and distributive exoribonuclease activities of the nuclear RNA exosome to protect stable RNA from degradation while selectively targeting and degrading less stable RNA. Substrate discrimination is lost when the distributive exoribonuclease activity of Rrp6 is inactivated, leading to degradation of stable and unstable RNA species. These data support a proofreading mechanism in which deadenylation by Rrp6 competes with Mtr4-dependent degradation to protect stable RNA while selectively targeting and degrading unstable RNA.

The RNA degradation pathway is involved in PPARα-modulated anti-oral tumorigenesis

Background: The activation of peroxisome proliferator-activated receptor alpha (PPARα) has been shown to reprogram tumor metabolism and exhibits great potential for treating anti-oral tumorigenesis. Methods: In this study, we used a pathway-based strategy to explore possible functional pathways involved in the anticancer activity of PPARα in oral cancer cells through next-generation sequencing (NGS) and bioinformatic approaches. Results: We found that 3919 genes were upregulated and 1060 genes were downregulated through PPARα activation. These genes were mainly involved in the proteasomal, mRNA surveillance, spliceosomal, RNA transport, and RNA degradation pathways, as indicated by GO and KEGG enrichment analysis. Importantly, a total of 13 upregulated genes in the RNA degradation pathway were identified including 3 core exosome factor genes (RRP43, RRP42, and CSL4), 2 TRAMP complex genes (TRF4 and Mtr4), 2 exosome cofactor genes (RRP6 and MPP6), 2 CCR4-NOT complex genes (CNOT2 and CNOT3), 2 Ski complex genes (SKI2 and Ski3), 1 decapping complex gene (EDC4), and 1 gene involved in 5’ exoribonuclease activity (XRN1). Conclusion: Our findings suggest that the activation of PPARα to upregulate the RNA degradation pathway might provide a new strategy for oral cancer treatment.

Exosome substrate targeting: the long and short of it

The exosome ribonuclease complex functions in both the limited trimming of the 3′-ends of nuclear substrates during RNA processing events and the complete destruction of nuclear and cytoplasmic RNAs. The two RNases of the eukaryotic exosome, Rrp44 (rRNA-processing protein 44) and Rrp6, are bound at either end of a catalytically inert cylindrical core. RNA substrates are threaded through the internal channel of the core to Rrp44 by RNA helicase components of the nuclear TRAMP complex (Trf4–Air2–Mtr4 polyadenylation complex) or the cytoplasmic Ski (superkiller) complex. Recent studies reveal that Rrp44 can also associate directly with substrates via channel-independent routes. Although the substrates of the exosome are known, it is not clear whether specific substrates are restricted to one or other pathway. Data currently available support the model that processed substrates are targeted directly to the catalytic subunits, whereas at least some substrates that are directed towards discard pathways must be threaded through the exosome core.