The general transcription factor IIH (TFIIH) is a multi-subunit protein complex involved in several fundamental biological processes, including transcription, DNA repair and cell cycle progression. TFIIH is organized in two functional subcomplexes, the core-TFIIH and the CDK-activating kinase (CAK), bridged together by the XPD subunit. XPD modulates the associated and dissociated state of the two sub-complexes and thereby it regulates the functionality of TFIIH and that of the two sub-complexes. Mutations in ERCC3/XPB, ERCC2/XPD or GTF2H5/TTDA genes, which encode distinct TFIIH subunits, lead to a range of autosomal recessive diseases, including the cancer prone xeroderma pigmentosum (XP) and the cancer-free multisystemic disorder trichothiodystrophy (TTD). Notably, XP and TTD may derive from distinct mutations in the same gene, either ERCC3/XPB or ERCC2/XPD. This thesis work is part of a large research project developed in the host laboratory whose aim is to investigate the altered molecular pathways that can explain the different cancer proneness of XP and TTD. Previously performed proteomic analysis allowed the identification of novel TFIIH interactors, including the ATP-dependent RNA helicase DEAD-box protein 1 (DDX1). Similarly to most DEAD-box proteins, DDX1 plays various functions in RNA metabolism, ranging from mRNA transcription and splicing to mRNA transport, translation and decay. Moreover, DDX1 is involved in the resolution of RNA:DNA hybrid molecules. Previous data from the host laboratory showed that DDX1 displays a stronger TFIIH interaction in TTD compared to XP and control primary dermal fibroblasts. The present study is focused on characterizing the biological function of TFIIH-DDX1 interaction under physiological or pathological conditions caused by TFIIH alterations. To achieve this goal, we investigate the relevance of DDX1 in two distinct pathways involving the action of TFIIH complex: the RNA polymerase II (RNPII)-mediated transcription and the cell cycle progression. After knocking down the expression of DDX1 by treating human cells with small interfering RNA (siRNA) molecules, we observe a significant reduction of RNPII and specific cyclin protein amounts, indicating alterations at the level of mRNA transcription and cell cycle progression, respectively. Considering the role of DDX1 as an RNA helicase involved in the unwinding of RNA:DNA hybrids, we then investigate whether DDX1 plays a role in the resolution of R-loops, three stranded hybrid nucleic acid structures that form during transcription and whose accumulation is associated with genome instability. After interference of DDX1 gene expression, a significant accumulation of R-loops is observed in human cells suggesting a link between the increased R-loop structures and the RNPII impairment. Finally, we investigate the amount of R-loops in cells from TTD or XP patients who carry alterations in the XPD subunit of the TFIIH complex. Notably, we observe a slight accumulation of R-loops in TTD but not in XP or control cells. Overall, these results highlight the relevance of TFIIH-DDX1 interaction in cell processes involving TFIIH activity and represent an interesting starting point for more in-depth analysis directed to identify the molecular basis underlying the different cancer predisposition in TTD and XP patients.
Caratterizzazione della funzione biologica di un nuovo interattore per il fattore di trascrizione IIH (TFIIH)
Characterizing the biological function of a novel interactor for transcription factor IIH (TFIIH)
CASTELLI, SARA
2020/2021
Abstract
The general transcription factor IIH (TFIIH) is a multi-subunit protein complex involved in several fundamental biological processes, including transcription, DNA repair and cell cycle progression. TFIIH is organized in two functional subcomplexes, the core-TFIIH and the CDK-activating kinase (CAK), bridged together by the XPD subunit. XPD modulates the associated and dissociated state of the two sub-complexes and thereby it regulates the functionality of TFIIH and that of the two sub-complexes. Mutations in ERCC3/XPB, ERCC2/XPD or GTF2H5/TTDA genes, which encode distinct TFIIH subunits, lead to a range of autosomal recessive diseases, including the cancer prone xeroderma pigmentosum (XP) and the cancer-free multisystemic disorder trichothiodystrophy (TTD). Notably, XP and TTD may derive from distinct mutations in the same gene, either ERCC3/XPB or ERCC2/XPD. This thesis work is part of a large research project developed in the host laboratory whose aim is to investigate the altered molecular pathways that can explain the different cancer proneness of XP and TTD. Previously performed proteomic analysis allowed the identification of novel TFIIH interactors, including the ATP-dependent RNA helicase DEAD-box protein 1 (DDX1). Similarly to most DEAD-box proteins, DDX1 plays various functions in RNA metabolism, ranging from mRNA transcription and splicing to mRNA transport, translation and decay. Moreover, DDX1 is involved in the resolution of RNA:DNA hybrid molecules. Previous data from the host laboratory showed that DDX1 displays a stronger TFIIH interaction in TTD compared to XP and control primary dermal fibroblasts. The present study is focused on characterizing the biological function of TFIIH-DDX1 interaction under physiological or pathological conditions caused by TFIIH alterations. To achieve this goal, we investigate the relevance of DDX1 in two distinct pathways involving the action of TFIIH complex: the RNA polymerase II (RNPII)-mediated transcription and the cell cycle progression. After knocking down the expression of DDX1 by treating human cells with small interfering RNA (siRNA) molecules, we observe a significant reduction of RNPII and specific cyclin protein amounts, indicating alterations at the level of mRNA transcription and cell cycle progression, respectively. Considering the role of DDX1 as an RNA helicase involved in the unwinding of RNA:DNA hybrids, we then investigate whether DDX1 plays a role in the resolution of R-loops, three stranded hybrid nucleic acid structures that form during transcription and whose accumulation is associated with genome instability. After interference of DDX1 gene expression, a significant accumulation of R-loops is observed in human cells suggesting a link between the increased R-loop structures and the RNPII impairment. Finally, we investigate the amount of R-loops in cells from TTD or XP patients who carry alterations in the XPD subunit of the TFIIH complex. Notably, we observe a slight accumulation of R-loops in TTD but not in XP or control cells. Overall, these results highlight the relevance of TFIIH-DDX1 interaction in cell processes involving TFIIH activity and represent an interesting starting point for more in-depth analysis directed to identify the molecular basis underlying the different cancer predisposition in TTD and XP patients.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/13225