ABSTRACT
Tudor domain containing protein 9 (TDRD9) is a RNA helicase normally expressed in the germline, where it is involved in the biosynthesis of PIWI-interacting RNAs (piRNAs). Here, we show that TDRD9 is highly expressed in a subset of non-small cell lung carcinomas and derived cell lines by hypomethylation of its CpG island. Furthermore, TDRD9 expression is associated with poor prognosis in lung adenocarcinoma. We find that downregulation of TDRD9 expression in TDRD9-positive cell lines causes a decrease in cell proliferation, S-phase cell cycle arrest, and apoptosis. Transcriptomic analysis demonstrated that TDRD9 knockdown causes upregulation of cell cycle and DNA repair genes. We also observed that TDRD9 knockdown triggers activation of the catalytic subunit of the DNA dependent protein kinase (DNA-PKcs) and phosphorylation of H2A.X, which are indicative of an increase of DNA double strand breaks. TDRD9-silenced cells also presented aberrant mitosis and abnormal-shaped nuclei indicating defects in chromosomal segregation. Finally, TDRD9 silencing caused hypersensitivity to the replication stress inducer aphidicolin, while overexpression of the protein increased resistance to the drug, suggesting that TDRD9 protects from replicative stress to TDRD9-positive tumor cells. Thus, our results place TDRD9 as a marker for prognosis and as a potential therapeutic target in a subset of lung carcinomas.
ABSTRACT
RNA polymerase II (RNAPII) transcription elongation is a highly regulated process that greatly influences mRNA levels as well as pre-mRNA splicing. Despite many studies in vitro, how chromatin modulates RNAPII elongation in vivo is still unclear. Here, we show that a decrease in the level of available canonical histones leads to more accessible chromatin with decreased levels of canonical histones and variants H2A.X and H2A.Z and increased levels of H3.3. With this altered chromatin structure, the RNAPII elongation rate increases, and the kinetics of pre-mRNA splicing is delayed with respect to RNAPII elongation. Consistent with the kinetic model of cotranscriptional splicing, the rapid RNAPII elongation induced by histone depletion promotes the skipping of variable exons in the CD44 gene. Indeed, a slowly elongating mutant of RNAPII was able to rescue this defect, indicating that the defective splicing induced by histone depletion is a direct consequence of the increased elongation rate. In addition, genome-wide analysis evidenced that histone reduction promotes widespread alterations in pre-mRNA processing, including intron retention and changes in alternative splicing. Our data demonstrate that pre-mRNA splicing may be regulated by chromatin structure through the modulation of the RNAPII elongation rate.
