Contribución de prefoldina a la expresión génica en células humanas

La prefoldina es una cochaperona que está presente en todos los organismos eucarióticos. Es conocida principalmente por su importante función en la dinámica del citoesqueleto, al favorecer el plegamiento de los monómeros de actina y tubulina durante su ensamblaje en filamentos de actina y microtúbulos, respectivamente. Adicionalmente, diferentes subunidades de este complejo heterohexamérico han sido encontradas en el núcleo y relacionadas con procesos nucleares en levaduras, plantas y metazoos. En esta tesis nos hemos propuesto estudiar qué papel desempeña la prefoldina en la expresión génica en células humanas. Para ello, hemos realizado un ensayo de RNA-seq que nos permitiera analizar qué efectos ocasionaba la deficiencia de las subunidades PFDN2 y PFDN5 en el transcriptoma de las células, bajo diferentes condiciones nutricionales. Los resultados mostraron que la deficiencia de estas dos subunidades afectó a la expresión de multitud de genes, siendo el efecto de PFDN5 más severo que el de PFDN2. Una parte de estos defectos puede ser explicado como consecuencia indirecta del papel de la prefoldina en la dinámica del citoesqueleto. Otros, pueden explicarse por la conocida función de PFDN5 como co-represor del protoncogén c-Myc. Además de estos efectos predecibles según lo conocido previamente, en nuestros resultados encontramos evidencias de la participación de estas subunidades en otros fenómenos. Por ejemplo, hemos encontrado un efecto de PFDN5 sobre la expresión del gen CDKN1A, que codifica para p21, un represor de la progresión del ciclo celular. Además, hemos detectado que PFDN5 se localiza físicamente sobre dicho gen. Este resultado nos permite explicar su influencia sobre la expresión de genes que se expresan cíclicamente, como los que codifican histonas. Asimismo, hemos detectado que las células deficientes en PFDN5 presentan deficiencias en la expresión de genes en función de sus características físicas, no funcionales, como su longitud o el número de intrones que presentan. También observamos que la deficiencia de PFDN5, y en menor medida la de PFDN2, empeoró de forma generalizada la eficiencia del procesamiento de intrones en el pre-mRNA, especialmente en los transcritos inmaduros de los genes que se expresan más. Igualmente hemos comprobado que las células deficientes en PFDN5 mostraron defectos en el acoplamiento temporal entre splicing y elongación de la transcripción, así como una disminución drástica de los niveles de fosforilación de la RNA polimerasa II en los residuos Ser2 de su dominio CTD. Los genes que muestran estas deficiencias en ausencia de PFDN5 presentan ocupación de esta proteína a lo largo de su región transcrita. En conjunto, nuestros resultados apoyan que la prefoldina contribuye de forma general al proceso de expresión génica en células humanas, al margen de los efectos regulatorios que sus otras funciones celulares puedan causar indirectamente

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

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-Agencia Estatal de Investigación [BFU2016-77728-C3-1-P to S.C. and BFU2017-85420-R to J.C.R.] co-financed with European Union funds (FEDER); Andalusian Government [P12-BIO1938MO, BIO271, US-1256285 to S.C., BIO321 to J.C.R.]; Junta de Andalucía (to L.P.-B.). Funding for open access charge: Ministerio de Ciencia e Innovación-Agencia Estatal de Investigación [BFU2016-77728-C3-1-P]

Human prefoldin modulates co-transcriptional pre-mRNA splicing

Trabajo presentado en el IV Meeting RNALife, celebrado en Sevilla (España) del 12 al 13 de julio de 2021.Prefoldin is a heterohexameric complex conserved from archaea to humans that plays a co-chaperone 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

Exploring the contribution of human prefoldin to gene expression

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.Prefoldin is a heterohexameric complex composed of six different subunits (PFDN1 to PFDN6) present from archaea to higher eukaryotes. It is a co-chaperone that cooperates with the cytoplasmic chaperonin CCT in the folding of actin and tubulin monomers. Eukaryotes also display a prefoldin-like complex, which contains PFDN2 and PFDN6 in addition to other non-canonical prefoldin subunits.We have previously showed that prefoldin is also localized in the nucleus of the yeast Saccharomyces cerevisiae where it plays a role in chromatin dynamics during transcription elongation (Millán-Zambrano, et al., 2013). We have also demonstrated that defective histone dynamics provokes changes in transcription elongation and co-transcriptional splicing in human cells (Jimeno-González et al., 2015). Here, we report that depletion of the canonical complex-specific PFDN5 subunit caused co-transcriptional splicing defects in CTNNBL1, a long human gene commonly used to analyze transcription elongation. However, we did not detect major alteration in the transcription rate of RNA polymerase II or its processivity (indirectly measured by pre-mRNA appearance) when PFDN5 was depleted. On the whole, these results suggest a possible role of prefoldin in human gene expression by contributing to co-transcriptional events.Peer reviewe

Defective histone supply causes changes in RNA polymerase II elongation rate and co-transcriptional 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.This work was funded by Spanish Ministry of Economy and Competitiveness (MINECO) Grants BFU-2011-23442 and BFU2014-53543-P (to J.C.R.) and Andalusian Government Grant P12CT52270 (to F.P.). S.J.-G. and A.M.M.-C. were supported by Juan de la Cierva Grants from MINECO. L.P.-B. was supported by the Asociación Española Contra el Cáncer

Prefoldin overexpression associates with the risk of mortality and metastasis in lung cancer

Resumen del trabajo presentado en el Meeting of the European Society for Radiotherapy and Oncology (ESTRO 2020), celebrado online del 28 de noviembre al 1 de diciembrePurpose or Objective Prefoldin (PFDN) is a co-chaperone that contributes to both cytoplasmic and nuclear biological processes. Canonical PFDN has a heterohexameric jellyfish-like structure. Four ß-type subunits (PFDN1, 2, 4 and 6) form two dimers onto two subunits of the α type (PFDN3 and 5). PFDN2 and 6 are also components of the URI-prefoldin-like complex, which has been described to promote cancer. It has been shown that PFDN1 overexpresion promotes epithelial-mesenchymal transition (EMT) and lung cancer (LC) progression in different LC cell lines and murine models whereas cyclin A knockdown alone induces EMT and increases cell migration and invasion ability. We investigated whether this putative involvement of canonical PFDN in LC translates into the clinic. Material and Methods 58 non-small cell LC patients with available tumor tissue samples (59% squamous and 41% adenocarcinoma) were assessed. The stages were as follows: 24% I, 7% II, 61% III, and 8% IV. 90% of patients were primarily treated with surgery and 69% received chemotherapy. 86% underwent thoracic radiotherapy either primarily (41%) or after locorregional recurrence (45%). The levels of PFDN1, 3, 5 were examined by immunoblotting. Additionally, the mRNA expression of 518 LC cases from The Cancer Genome Atlas (TCGA) database was evaluated. To assess the risk of mortality and recurrences we used Kaplan-Meier and Cox proportional hazards analysesResults PFDN1, 3, 5 and cyclin A overexpression (+++) were found in 22 (38%), 31 (53%), 24 (41%), and 14 (24%) tumor samples. After a follow up of 40 months, 39 (67%) patients were alive and 34 (58%) had experienced a recurrence (24 were distant metastasis). Body surface area and stage associated with overall survival (OS; p=0.01 and p=0.036, respectively), disease-free survival (DFS; p=0.033 and p=0.038, respectively), and distant metastasis-free survival (DMFS; p=0.002 and p=0.025, respectively) in the univariate analysis. In addition, the use of radiotherapy and chemotherapy also associated with DMFS (p=0.005 and p=0.015, respectively). PFDN1, 3 and 5 overexpression were associated with lower OS (p=0.002, p=0.015, and p=0.002, respectively), lower DFS (p=0.01, p=0.042, and p=0.055, respectively), and lower DMFS (p=0.011, p=0.036, and p=0.11, respectively). There was not any association with local recurrence. In the multivariate analysis, the PFDN5 retained significance for OS (HR 5.09; p=0.007) and the PFDN1 for DFS (HR 5.15; p=0.01) and DMFS (HR 5.45; p=0.05). In the TCGA adenocarcinoma cohort, there was a high correlation between PFDN1 and 5 (Pearson coefficient: 0.53; p <0.0001), a high mRNA expression of PFDN3 in the tumor compare with the normal tissue (p <0.0001), and PFDN1 overexpression showed lower OS (p=0.034). Conclusion Overexpression of canonical PFDN associates with the risk of mortality and metastasis in non-small cell LC. These response markers may be usefull biomarkers for guiding therapy intensity in an individualized therapy.Peer reviewe

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.This work was co-funded by the Spanish Ministry of Economy and Competitiveness (BFU2017-85420-R to JCR and BFU2016-7772-C3-1-P to SC) and by the Andalusian Government (BIO-271) and the University of Seville (US-1256285 to SC) in cooperation with the European Regional Development Fund.Peer reviewe