Ubiquitin-Proteasome System Regulation of an Evolutionarily Conserved RNA Polymerase II-Associated Factor 1 Involved in Pancreatic Oncogenesis.

The evolutionarily conserved RNA polymerase II-associated factor 1 (Paf1) from yeast to humans regulates transcription and associated processes, and thus, malfunctions and/or misregulations of Paf1 are associated with cellular pathologies. Indeed, Paf1 (also known as PD2 or pancreatic differentiation 2) is found to be upregulated in poorly differentiated cancer cells, and such upregulation is involved in cellular transformation or oncogenesis. However, the basis for Paf1 upregulation in these cells remains largely unknown. In light of this, we have tested here the idea that the ubiquitin-proteasome system (UPS) regulates the cellular abundance of Paf1. In this direction, we analyzed the role of UPS in regulation of Paf1's abundance in yeast. We find that Paf1 undergoes ubiquitylation and is degraded by the 26S proteasome in yeast, thus deciphering UPS regulation of an evolutionarily conserved factor, Paf1, involved in various cellular processes at the crossroads of the cancer networks. Likewise, Paf1 undergoes proteasomal degradation in well-differentiated, but not poorly differentiated, pancreatic cancer cells, hence pointing to the UPS in upregulation of Paf1 in poorly differentiated cancers. Collectively, our results reveal UPS regulation of Paf1 and suggest downregulation of UPS in elevating Paf1's abundance in poorly differentiated cancers.

Regulation of eukaryotic transcription initiation in response to cellular stress.

Transcription initiation is a vital step in the regulation of eukaryotic gene expression. It can be dysregulated in response to various cellular stressors which is associated with numerous human diseases including cancer. Transcription initiation is facilitated via many gene-specific trans-regulatory elements such as transcription factors, activators, and coactivators through their interactions with transcription pre-initiation complex (PIC). These trans-regulatory elements can uniquely facilitate PIC formation (hence, transcription initiation) in response to cellular nutrient stress. Cellular nutrient stress also regulates the activity of other pathways such as target of rapamycin (TOR) pathway. TOR pathway exhibits distinct regulatory mechanisms of transcriptional activation in response to stress. Like TOR pathway, the cell cycle regulatory pathway is also found to be linked to transcriptional regulation in response to cellular stress. Several transcription factors such as p53, C/EBP Homologous Protein (CHOP), activating transcription factor 6 (ATF6α), E2F, transforming growth factor (TGF)-ß, Adenomatous polyposis coli (APC), SMAD, and MYC have been implicated in regulation of transcription of target genes involved in cell cycle progression, apoptosis, and DNA damage repair pathways. Additionally, cellular metabolic and oxidative stressors have been found to regulate the activity of long non-coding RNAs (lncRNA). LncRNA regulates transcription by upregulating or downregulating the transcription regulatory proteins involved in metabolic and cell signaling pathways. Numerous human diseases, triggered by chronic cellular stressors, are associated with abnormal regulation of transcription. Hence, understanding these mechanisms would help unravel the molecular regulatory insights with potential therapeutic interventions. Therefore, here we emphasize the recent advances of regulation of eukaryotic transcription initiation in response to cellular stress.

SARS-CoV-2 and UPS with potentials for therapeutic interventions.

The Ubiquitin proteasome system (UPS), an essential eukaryotic/host/cellular post-translational modification (PTM), plays a critical role in the regulation of diverse cellular functions including regulation of protein stability, immune signaling, antiviral activity, as well as virus replication. Although UPS regulation of viral proteins may be utilized by the host as a defense mechanism to invade viruses, viruses may have adapted to take advantage of the host UPS. This system can be manipulated by viruses such as the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) to stimulate various steps of the viral replication cycle and facilitate pathogenesis, thereby causing the respiratory disease COVID-19. Many SARS-CoV-2 encoded proteins including open reading frame 3a (ORF3a), ORF6, ORF7a, ORF9b, and ORF10 interact with the host's UPS machinery, influencing host immune signaling and apoptosis. Moreover, SARS-CoV-2 encoded papain-like protease (PLpro) interferes with the host UPS to facilitate viral replication and to evade the host's immune system. These alterations in SARS-CoV-2 infected cells have been revealed by various proteomic studies, suggesting potential targets for clinical treatment. To provide insight into the underlying causes of COVID-19 and suggest possible directions for therapeutic interventions, this paper reviews the intricate relationship between SARS-CoV-2 and UPS. Promising treatment strategies are also investigated in this paper including targeting PLpro with zinc-ejector drugs, as well as targeting viral non-structural protein (nsp12) via heat treatment associated ubiquitin-mediated proteasomal degradation to reduce viral pathogenesis.

Ubiquitin-proteasome system regulation of a key gene regulatory factor, Paf1C.

Paf1 (Polymerase-associated factor 1) complex (Paf1C) is evolutionarily conserved from yeast to humans, and facilitates transcription elongation as well as co-transcriptional histone covalent modifications and mRNA 3'-end processing. Thus, Paf1C is a key player in regulation of eukaryotic gene expression. Paf1C consists of Paf1, Cdc73, Ctr9, Leo1 and Rtf1 in both yeast and humans, but it has an additional component, Ski8, in humans. The abundances of these components regulate the assembly of Paf1C and/or its functions, thus implying the mechanisms involved in regulating the abundances of the Paf1C components in altered gene expression and hence cellular pathologies. Towards finding the mechanisms associated with the abundances of the Paf1C components, we analyzed here whether the Paf1C components are regulated via targeted ubiquitylation and 26S proteasomal degradation. We find that the Paf1C components except Paf1 do not undergo the 26S proteasomal degradation in both yeast and humans. Paf1 is found to be regulated by the ubiquitin-proteasome system (UPS) in yeast and humans. Alteration of such regulation changes Paf1's abundance, leading to aberrant gene expression. Intriguingly, while the Rtf1 component of Paf1C does not undergo the 26S proteasomal degradation, it is found to be ubiquitylated, suggesting that Rtf1 ubiquitylation could be engaged in Paf1C assembly and/or functions. Collectively, our results reveal distinct UPS regulation of the Paf1C components, Paf1 and Rtf1, in a proteolysis-dependent and -independent manners, respectively, with functional implications.

A novel ubiquitin-proteasome system regulation of Sgf73/ataxin-7 that maintains the integrity of the coactivator SAGA in orchestrating transcription.

Ataxin-7 maintains the integrity of Spt-Ada-Gcn5-Acetyltransferase (SAGA), an evolutionarily conserved coactivator in stimulating preinitiation complex (PIC) formation for transcription initiation, and thus, its upregulation or downregulation is associated with various diseases. However, it remains unknown how ataxin-7 is regulated that could provide new insights into disease pathogenesis and therapeutic interventions. Here, we show that ataxin-7's yeast homologue, Sgf73, undergoes ubiquitylation and proteasomal degradation. Impairment of such regulation increases Sgf73's abundance, which enhances recruitment of TATA box-binding protein (TBP) (that nucleates PIC formation) to the promoter but impairs transcription elongation. Further, decreased Sgf73 level reduces PIC formation and transcription. Thus, Sgf73 is fine-tuned by ubiquitin-proteasome system (UPS) in orchestrating transcription. Likewise, ataxin-7 undergoes ubiquitylation and proteasomal degradation, alteration of which changes ataxin-7's abundance that is associated with altered transcription and cellular pathologies/diseases. Collectively, our results unveil a novel UPS regulation of Sgf73/ataxin-7 for normal cellular health and implicate alteration of such regulation in diseases.

Genome-Wide Regulations of the Preinitiation Complex Formation and Elongating RNA Polymerase II by an E3 Ubiquitin Ligase, San1.

San1 ubiquitin ligase is involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and proteasomal degradation. Since several transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in transcription regulation. To address this, we analyzed the role of San1 in the genome-wide association of TATA box binding protein (TBP; which nucleates preinitiation complex [PIC] formation for transcription initiation) and RNA polymerase II (Pol II). Our results reveal the roles of San1 in regulating TBP recruitment to the promoters and Pol II association with the coding sequences and, hence, PIC formation and coordination of elongating Pol II, respectively. Consistently, transcription is altered in the absence of San1. Such transcriptional alteration is associated with impaired ubiquitylation and proteasomal degradation of Spt16 and gene association of Paf1 but not the incorporation of centromeric histone, Cse4, into the active genes in the Δsan1 strain. Collectively, our results demonstrate distinct functions of a nuclear protein quality control factor in regulating the genome-wide PIC formation and elongating Pol II (and hence transcription), thus unraveling new gene regulatory mechanisms.

An F-Box Protein, Mdm30, Interacts with TREX Subunit Sub2 To Regulate Cellular Abundance Cotranscriptionally in Orchestrating mRNA Export Independently of Splicing and Mitochondrial Function.

Although an F-box protein, Mdm30, is found to regulate ubiquitylation of the Sub2 component of TREX (transcription-export) complex for proteasomal degradation in stimulation of mRNA export, it remains unknown whether such ubiquitin-proteasome system (UPS) regulation of Sub2 occurs cotranscriptionally via its interaction with Mdm30. Further, it is unclear whether impaired UPS regulation of Sub2 in the absence of Mdm30 alters mRNA export via splicing defects of export factors and/or mitochondrial dynamics/function, since Sub2 controls mRNA splicing and Mdm30 regulates mitochondrial aggregation. Here, we show that Mdm30 interacts with Sub2, and temporary shutdown of Mdm30 enhances Sub2's abundance and impairs mRNA export. Likewise, Sub2's abundance is increased following transcriptional inhibition. These results support Mdm30's direct role in regulation of Sub2's cellular abundance in a transcription-dependent manner. Consistently, the chromatin-bound Sub2 level is increased in the absence of Mdm30. Further, we find that Mdm30 does not facilitate splicing of export factors. Moreover, Mdm30 does not have a dramatic effect on mitochondrial respiration/function, and mRNA export occurs in the absence of Fzo1, which is required for mitochondrial dynamics/respiration. Collective results reveal that Mdm30 interacts with Sub2 for proteasomal degradation in a transcription-dependent manner to promote mRNA export independently of splicing or mitochondrial function, thus advancing our understanding of mRNA export.

Distinct Functions of the Cap-Binding Complex in Stimulation of Nuclear mRNA Export.

Cap-binding complex (CBC) associates cotranscriptionally with the cap structure at the 5' end of nascent mRNA to protect it from exonucleolytic degradation. Here, we show that CBC promotes the targeting of an mRNA export adaptor, Yra1 (forming transcription export [TREX] complex with THO and Sub2), to the active genes and enhances mRNA export in Saccharomyces cerevisiae Likewise, recruitment of Npl3 (an hnRNP involved in mRNA export via formation of export-competent ribonuclear protein complex [RNP]) to the active genes is facilitated by CBC. Thus, CBC enhances targeting of the export factors and promotes mRNA export. Such function of CBC is not mediated via THO and Sub2 of TREX, cleavage and polyadenylation factors, or Sus1 (that regulates mRNA export via transcription export 2 [TREX-2]). However, CBC promotes splicing of SUS1 mRNA and, consequently, Sus1 protein level and mRNA export via TREX-2. Collectively, our results support the hypothesis that CBC promotes recruitment of Yra1 and Npl3 to the active genes, independently of THO, Sub2, or cleavage and polyadenylation factors, and enhances mRNA export via TREX and RNP, respectively, in addition to its role in facilitating SUS1 mRNA splicing to increase mRNA export through TREX-2, revealing distinct stimulatory functions of CBC in mRNA export.

Two Distinct Regulatory Mechanisms of Transcriptional Initiation in Response to Nutrient Signaling.

SAGA (Spt-Ada-Gcn5-Acetyltransferase) and TFIID (transcription factor IID) have been previously shown to facilitate the formation of the PIC (pre-initiation complex) at the promoters of two distinct sets of genes. Here, we demonstrate that TFIID and SAGA differentially participate in the stimulation of PIC formation (and hence transcriptional initiation) at the promoter of PHO84, a gene for the high-affinity inorganic phosphate (Pi) transporter for crucial cellular functions, in response to nutrient signaling. We show that transcriptional initiation of PHO84 occurs predominantly in a TFIID-dependent manner in the absence of Pi in the growth medium. Such TFIID dependency is mediated via the NuA4 (nucleosome acetyltransferase of H4) histone acetyltransferase (HAT). Intriguingly, transcriptional initiation of PHO84 also occurs in the presence of Pi in the growth medium, predominantly via the SAGA complex, but independently of NuA4 HAT. Thus, Pi in the growth medium switches transcriptional initiation of PHO84 from NuA4-TFIID to SAGA dependency. Further, we find that both NuA4-TFIID- and SAGA-dependent transcriptional initiations of PHO84 are facilitated by the 19S proteasome subcomplex or regulatory particle (RP) via enhanced recruitment of the coactivators SAGA and NuA4 HAT, which promote TFIID-independent and -dependent PIC formation for transcriptional initiation, respectively. NuA4 HAT does not regulate activator binding to PHO84, but rather facilitates PIC formation for transcriptional initiation in the absence of Pi in the growth medium. On the other hand, SAGA promotes activator recruitment to PHO84 for transcriptional initiation in the growth medium containing Pi. Collectively, our results demonstrate two distinct stimulatory pathways for PIC formation (and hence transcriptional initiation) at PHO84 by TFIID, SAGA, NuA4, and 19S RP in the presence and absence of an essential nutrient, Pi, in the growth media, thus providing new regulatory mechanisms of transcriptional initiation in response to nutrient signaling.

An mRNA Capping Enzyme Targets FACT to the Active Gene To Enhance the Engagement of RNA Polymerase II into Transcriptional Elongation.

We have recently demonstrated that an mRNA capping enzyme, Cet1, impairs promoter-proximal accumulation/pausing of RNA polymerase II (Pol II) independently of its capping activity in Saccharomyces cerevisiae to control transcription. However, it is still unknown how Pol II pausing is regulated by Cet1. Here, we show that Cet1's N-terminal domain (NTD) promotes the recruitment of FACT (facilitates chromatin transcription that enhances the engagement of Pol II into transcriptional elongation) to the coding sequence of an active gene, ADH1, independently of mRNA-capping activity. Absence of Cet1's NTD decreases FACT targeting to ADH1 and consequently reduces the engagement of Pol II in transcriptional elongation, leading to promoter-proximal accumulation of Pol II. Similar results were also observed at other genes. Consistently, Cet1 interacts with FACT. Collectively, our results support the notion that Cet1's NTD promotes FACT targeting to the active gene independently of mRNA-capping activity in facilitating Pol II's engagement in transcriptional elongation, thus deciphering a novel regulatory pathway of gene expression.

Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation.

FACT (facilitates chromatin transcription), an evolutionarily conserved histone chaperone involved in transcription and other DNA transactions, is upregulated in cancers, and its downregulation is associated with cellular death. However, it is not clearly understood how FACT is fine-tuned for normal cellular functions. Here, we show that the FACT subunit Spt16 is ubiquitylated by San1 (an E3 ubiquitin ligase) and degraded by the 26S proteasome. Enhanced abundance of Spt16 in the absence of San1 impairs transcriptional elongation. Likewise, decreased abundance of Spt16 also reduces transcription. Thus, an optimal level of Spt16 is required for efficient transcriptional elongation, which is maintained by San1 via ubiquitylation and proteasomal degradation. Consistently, San1 associates with the coding sequences of active genes to regulate Spt16's abundance. Further, we found that enhanced abundance of Spt16 in the absence of San1 impairs chromatin reassembly at the coding sequence, similarly to the results seen following inactivation of Spt16. Efficient chromatin reassembly enhances the fidelity of transcriptional elongation. Taken together, our results demonstrate for the first time a fine-tuning of FACT by a ubiquitin proteasome system in promoting chromatin reassembly in the wake of elongating RNA polymerase II and transcriptional elongation, thus revealing novel regulatory mechanisms of gene expression.

Regulation of Antisense Transcription by NuA4 Histone Acetyltransferase and Other Chromatin Regulatory Factors.

NuA4 histone lysine (K) acetyltransferase (KAT) promotes transcriptional initiation of TATA-binding protein (TBP)-associated factor (TAF)-dependent ribosomal protein genes. TAFs have also been recently found to enhance antisense transcription from the 3' end of the GAL10 coding sequence. However, it remains unknown whether, like sense transcription of the ribosomal protein genes, TAF-dependent antisense transcription of GAL10 also requires NuA4 KAT. Here, we show that NuA4 KAT associates with the GAL10 antisense transcription initiation site at the 3' end of the coding sequence. Such association of NuA4 KAT depends on the Reb1p-binding site that recruits Reb1p activator to the GAL10 antisense transcription initiation site. Targeted recruitment of NuA4 KAT to the GAL10 antisense transcription initiation site promotes GAL10 antisense transcription. Like NuA4 KAT, histone H3 K4/36 methyltransferases and histone H2B ubiquitin conjugase facilitate GAL10 antisense transcription, while the Swi/Snf and SAGA chromatin remodeling/modification factors are dispensable for antisense, but not sense, transcription of GAL10. Taken together, our results demonstrate for the first time the roles of NuA4 KAT and other chromatin regulatory factors in controlling antisense transcription, thus illuminating chromatin regulation of antisense transcription.