Overexpression of canonical prefoldin associates with the risk of mortality and metastasis in non-small cell lung cancer

Canonical prefoldin is a protein cochaperone composed of six di erent subunits (PFDN1 to 6). PFDN1 overexpression promotes epithelial–mesenchymal transition (EMT) and increases the growth of xenograft lung cancer (LC) cell lines. We investigated whether this putative involvement of canonical PFDN in LC translates into the clinic. First, the mRNA expression of 518 non-small cell LC (NSCLC) cases from The Cancer Genome Atlas (TCGA) database was evaluated. Patients with PFDN1 overexpression had lower overall survival (OS; 45 vs. 86 months; p = 0.034). We then assessed the impact of PFDN expression on outcome in 58 NSCLC patients with available tumor tissue samples. PFDN1, 3, and 5 overexpression were found in 38% (n = 22), 53% (n = 31), and 41% (n = 24) of tumor samples. PFDN1, 3, and 5 overexpression were significantly associated with lower OS, lower disease-free survival (DFS), and lower distant metastasis-free survival (DMFS) for PFDN1 and 3 with a trend for PFDN5. In multivariate analysis, PFDN5 retained significance for OS (hazard ratio (HR) 2.56; p = 0.007) and PFDN1 for DFS (HR 2.53; p = 0.010) and marginally for DMFS (HR 2.32; p = 0.053). Our results indicate that protein response markers, such as PFDN1, 3, and 5, may complement mRNA signatures and be useful for determining the most appropriate therapy for NSCLC patients

One step back before moving forward: regulation of transcription elongation by arrest and backtracking

RNA polymerase II backtracking is a well-known phenomenon, but its involvement in gene regulation is yet to be addressed. Structural studies into the backtracked complex, new reactivation mechanisms and genome-wide approaches are shedding some light on this interesting aspect of gene transcription. In this review, we briefly summarise these new findings, comment about some results recently obtained in our laboratory, and propose a new model for the influence of the chromatin context on RNA polymerase II backtracking.Ministerio de Economía y Competitividad de España. BFU2007-67575-C03-02 y BFU-2010-21975-C03-03Junta de Andalucía. P07-CVI-02623 y P08-CVI-0350

Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration

mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5ʹ exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3ʹ was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favouring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.Ministerio de Economía y Competitividad BFU2016-77728- C3-1-P, BFU2016-77728-C3-3-P, BFU2016- 77728-C3-2-P, RED2018-102467-TJunta de Andalucía BIO271, US-1256285, BIO258, UJA 1260360Generalitat Valenciana AICO/2019/08

Human prefoldin modulates co-transcriptional pre-mRNA splicing

Prefoldin is a heterohexameric complex conserved from archaea to humans that plays a cochaperone role during the co-translational folding of actin and tubulin monomers. Additional functions of prefoldin have been described, including a positive contribution to transcription elongation and chromatin dynamics in yeast. Here we show that prefoldin perturbations provoked transcriptional alterations across the human genome. Severe pre-mRNA splicing defects were also detected, particularly after serum stimulation. We found impairment of co-transcriptional splicing during transcription elongation, which explains why the induction of long genes with a high number of introns was affected the most. We detected genome-wide prefoldin binding to transcribed genes and found that it correlated with the negative impact of prefoldin depletion on gene expression. Lack of prefoldin caused global decrease in Ser2 and Ser5 phosphorylation of the RNA polymerase II carboxy-terminal domain. It also reduced the recruitment of the CTD kinase CDK9 to transcribed genes, and the association of splicing factors PRP19 and U2AF65 to chromatin, which is known to depend on CTD phosphorylation. Altogether the reported results indicate that human prefoldin is able to act locally on the genome to modulate gene expression by influencing phosphorylation of elongating RNA polymerase II, and thereby regulating co-transcriptional splicing.Ministerio de Ciencia e Innovación BFU2016-77728-C3-1-P, BFU2017-85420-RJunta de Andalucía P12- BIO1938MO, BIO271, US-1256285, BIO32