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.

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 different 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.

Functional Contributions of Prefoldin to Gene Expression.

Prefoldin is a co-chaperone that evolutionarily originates in archaea, is universally present in all eukaryotes and acts as a co-chaperone by facilitating the supply of unfolded or partially folded substrates to class II chaperonins. Eukaryotic prefoldin is known mainly for its functional relevance in the cytoplasmic folding of actin and tubulin monomers during cytoskeleton assembly. However, the role of prefoldin in chaperonin-mediated folding is not restricted to cytoskeleton components, but extends to both the assembly of other cytoplasmic complexes and the maintenance of functional proteins by avoiding protein aggregation and facilitating proteolytic degradation. Evolution has favoured the diversification of prefoldin subunits, and has allowed the so-called prefoldin-like complex, with specialised functions, to appear. Subunits of both canonical and prefoldin-like complexes have also been found in the nucleus of yeast and metazoan cells, where they have been functionally connected with different gene expression steps. Plant prefoldin has also been detected in the nucleus and is physically associated with a gene regulator. Here we summarise information available on the functional involvement of prefoldin in gene expression, and discuss the implications of these results for the relationship between prefoldin structure and function.

Defective histone supply causes changes in RNA polymerase II elongation rate and cotranscriptional pre-mRNA splicing.

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.