Transcription elongation mechanisms of RNA polymerases I, II, and III and their therapeutic implications.

Transcription is a tightly regulated, complex, and essential cellular process in all living organisms. Transcription is comprised of three steps, transcription initiation, elongation, and termination. The distinct transcription initiation and termination mechanisms of eukaryotic RNA polymerases I, II, and III (Pols I, II, and III) have long been appreciated. Recent methodological advances have empowered high-resolution investigations of the Pols' transcription elongation mechanisms. Here, we review the kinetic similarities and differences in the individual steps of Pol I-, II-, and III-catalyzed transcription elongation, including NTP binding, bond formation, pyrophosphate release, and translocation. This review serves as an important summation of Saccharomyces cerevisiae (yeast) Pol I, II, and III kinetic investigations which reveal that transcription elongation by the Pols is governed by distinct mechanisms. Further, these studies illustrate how basic, biochemical investigations of the Pols can empower the development of chemotherapeutic compounds.

TBP facilitates RNA Polymerase I transcription following mitosis.

The TATA-box binding protein (TBP) is the sole transcription factor common in the initiation complexes of the three major eukaryotic RNA Polymerases (Pol I, II and III). Although TBP is central to transcription by the three RNA Pols in various species, the emergence of TBP paralogs throughout evolution has expanded the complexity in transcription initiation. Furthermore, recent studies have emerged that questioned the centrality of TBP in mammalian cells, particularly in Pol II transcription, but the role of TBP and its paralogs in Pol I transcription remains to be re-evaluated. In this report, we show that in murine embryonic stem cells TBP localizes onto Pol I promoters, whereas the TBP paralog TRF2 only weakly associates to the Spacer Promoter of rDNA, suggesting that it may not be able to replace TBP for Pol I transcription. Importantly, acute TBP depletion does not fully disrupt Pol I occupancy or activity on ribosomal RNA genes, but TBP binding in mitosis leads to efficient Pol I reactivation following cell division. These findings provide a more nuanced role for TBP in Pol I transcription in murine embryonic stem cells.

Hmo1 Promotes Efficient Transcription Elongation by RNA Polymerase I in Saccharomyces cerevisiae.

RNA polymerase I (Pol I) is responsible for synthesizing the three largest eukaryotic ribosomal RNAs (rRNAs), which form the backbone of the ribosome. Transcription by Pol I is required for cell growth and, therefore, is subject to complex and intricate regulatory mechanisms. To accomplish this robust regulation, the cell engages a series of trans-acting transcription factors. One such factor, high mobility group protein 1 (Hmo1), has long been established as a trans-acting factor for Pol I in Saccharomyces cerevisiae; however, the mechanism by which Hmo1 promotes rRNA synthesis has not been defined. Here, we investigated the effect of the deletion of HMO1 on transcription elongation by Pol I in vivo. We determined that Hmo1 is an important activator of transcription elongation, and without this protein, Pol I accumulates across rDNA in a sequence-specific manner. Our results demonstrate that Hmo1 promotes efficient transcription elongation by rendering Pol I less sensitive to pausing in the G-rich regions of rDNA.

Global kinetic mechanism describing single nucleotide incorporation for RNA polymerase I reveals fast UMP incorporation.

RNA polymerase I (Pol I) is responsible for synthesizing ribosomal RNA, which is the rate limiting step in ribosome biogenesis. We have reported wide variability in the magnitude of the rate constants defining the rate limiting step in sequential nucleotide additions catalyzed by Pol I. in this study we sought to determine if base identity impacts the rate limiting step of nucleotide addition catalyzed by Pol I. To this end, we report a transient state kinetic interrogation of AMP, CMP, GMP, and UMP incorporations catalyzed by Pol I. We found that Pol I uses one kinetic mechanism to incorporate all nucleotides. However, we found that UMP incorporation is faster than AMP, CMP, and GMP additions. Further, we found that endonucleolytic removal of a dimer from the 3' end was fastest when the 3' terminal base is a UMP. It has been previously shown that both downstream and upstream template sequence identity impacts the kinetics of nucleotide addition. The results reported here show that the incoming base identity also impacts the magnitude of the observed rate limiting step.

Multiple myeloma: clinical characteristics, current therapies and emerging innovative treatments targeting ribosome biogenesis dynamics.

Multiple myeloma (MM) is a clinical disorder characterized by aberrant plasma cell growth in the bone marrow microenvironment. Globally, the prevalence of MM has been steadily increasing at an alarming rate. In the United States, more than 30,000 cases will be diagnosed in 2024 and it accounts for about 2% of cancer diagnoses and more than 2% of cancer deaths, more than double the worldwide figure. Both symptomatic and active MM are distinguished by uncontrolled plasma cell growth, which results in severe renal impairment, anemia, hypercalcemia, and bone loss. Multiple drugs have been approved by the FDA and are now widely used in clinical practice for MM. Although triplet and quadruplet induction regimens, autologous stem cell transplantation (ASCT), and maintenance treatment are used, MM continues to be an incurable illness characterized by relapses that may occur at various phases of its progression. MM patients with frailty, extramedullary disease, plasma cell leukemia, central nervous system recurrence, functional high risk, and the elderly are among those with the greatest current unmet needs. The high cost of care is an additional challenge. MM cells are highly protein secretary cells and thus are dependent on the activation of certain translation pathways. MM also has a high chance of altering ribosomal protein-encoding genes like MYC mutation. In this article we discuss the importance of ribosome biogenesis in promoting MM and RNA polymerase I inhibition as an upcoming treatment with potential promise for MM patients.

Targeting the ribosome to treat multiple myeloma.

The high rates of protein synthesis and processing render multiple myeloma (MM) cells vulnerable to perturbations in protein homeostasis. The induction of proteotoxic stress by targeting protein degradation with proteasome inhibitors (PIs) has revolutionized the treatment of MM. However, resistance to PIs is inevitable and represents an ongoing clinical challenge. Our first-in-human study of the selective inhibitor of RNA polymerase I transcription of ribosomal RNA genes, CX-5461, has demonstrated a potential signal for anti-tumor activity in three of six heavily pre-treated MM patients. Here, we show that CX-5461 has potent anti-myeloma activity in PI-resistant MM preclinical models in vitro and in vivo. In addition to inhibiting ribosome biogenesis, CX-5461 causes topoisomerase II trapping and replication-dependent DNA damage, leading to G2/M cell-cycle arrest and apoptotic cell death. Combining CX-5461 with PI does not further enhance the anti-myeloma activity of CX-5461 in vivo. In contrast, CX-5461 shows synergistic interaction with the histone deacetylase inhibitor panobinostat in both the Vk∗MYC and the 5T33-KaLwRij mouse models of MM by targeting ribosome biogenesis and protein synthesis through distinct mechanisms. Our findings thus provide strong evidence to facilitate the clinical development of targeting the ribosome to treat relapsed and refractory MM.

CX­5461 potentiates imatinib­induced apoptosis in K562 cells by stimulating KIF1B expression.

The selective RNA polymerase I inhibitor CX-5461 has been shown to be effective in treating some types of leukemic disorders. Emerging evidence suggests that combined treatments with CX-5461 and other chemotherapeutic agents may achieve enhanced effectiveness as compared with monotherapies. Currently, pharmacodynamic properties of the combination of CX-5461 with tyrosine kinase inhibitors remain to be explored. The present study tested whether CX-5461 could potentiate the effect of imatinib in the human chronic myeloid leukemia cell line K562, which is p53-deficient. It was demonstrated that CX-5461 at 100 nM, which was non-cytotoxic in K562 cells, potentiated the pro-apoptotic effect of imatinib. Mechanistically, the present study identified that the upregulated expression of kinesin family member 1B (KIF1B) gene might be involved in mediating the pro-apoptotic effect of imatinib/CX-5461 combination. Under the present experimental settings, however, neither CX-5461 nor imatinib alone exhibited a significant effect on KIF1B expression. Moreover, using other leukemic cell lines, it was demonstrated that regulation of KIF1B expression by imatinib/CX-5461 was not a ubiquitous phenomenon in leukemic cells and should be studied in a cell type-specific manner. In conclusion, the results suggested that the synergistic interaction between CX-5461 and imatinib may be of potential clinical value for the treatment of tyrosine kinase inhibitor-resistant chronic myeloid leukemia.

Inhibition of TAF1B impairs ribosome biosynthesis and suppresses cell proliferation in stomach adenocarcinoma through promoting c-MYC mRNA degradation.

Hyperactivation of ribosome biosynthesis (RiBi) is a hallmark of cancer, and targeting ribosome biogenesis has emerged as a potential therapeutic strategy. The depletion of TAF1B, a major component of selectivity factor 1 (SL1), disrupts the pre-initiation complex, preventing RNA polymerase I from binding ribosomal DNA and inhibiting the hyperactivation of RiBi. Here, we investigate the role of TAF1B, in regulating RiBi and proliferation in stomach adenocarcinoma (STAD). We disclosed that the overexpression of TAF1B correlates with poor prognosis in STAD, and found that knocking down TAF1B effectively inhibits STAD cell proliferation and survival in vitro and in vivo. TAF1B knockdown may also induce nucleolar stress, and promote c-MYC degradation in STAD cells. Furthermore, we demonstrate that TAF1B depletion impairs rRNA gene transcription and processing, leading to reduced ribosome biogenesis. Collectively, our findings suggest that TAF1B may serve as a potential therapeutic target for STAD and highlight the importance of RiBi in cancer progression.

Reverse Genetics of Hepatitis C Virus Using an RNA Polymerase I-Mediated Transcription.

The reverse genetics system commonly used for the production of hepatitis C virus (HCV), which is a major causative agent of liver diseases, involves introduction of the viral genomic RNA synthesized in vitro into human hepatoma cells by electroporation. As an alternative methodology, we describe a cell culture system based on transfection with an expression plasmid containing a full-length HCV cDNA clone flanked by RNA polymerase I promoter and terminator sequences to generate infectious virus particles from transfected cells.

VAV2 orchestrates the interplay between regenerative proliferation and ribogenesis in both keratinocytes and oral squamous cell carcinoma.

VAV2 is an activator of RHO GTPases that promotes and maintains regenerative proliferation-like states in normal keratinocytes and oral squamous cell carcinoma (OSCC) cells. Here, we demonstrate that VAV2 also regulates ribosome biogenesis in those cells, a program associated with poor prognosis of human papilloma virus-negative (HPV-) OSCC patients. Mechanistically, VAV2 regulates this process in a catalysis-dependent manner using a conserved pathway comprising the RAC1 and RHOA GTPases, the PAK and ROCK family kinases, and the c-MYC and YAP/TAZ transcription factors. This pathway directly promotes RNA polymerase I activity and synthesis of 47S pre-rRNA precursors. This process is further consolidated by the upregulation of ribosome biogenesis factors and the acquisition of the YAP/TAZ-dependent undifferentiated cell state. Finally, we show that RNA polymerase I is a therapeutic Achilles' heel for both keratinocytes and OSCC patient-derived cells endowed with high VAV2 catalytic activity. Collectively, these findings highlight the therapeutic potential of modulating VAV2 and the ribosome biogenesis pathways in both preneoplastic and late progression stages of OSCC.

Rescue of Infectious Salmon Anemia Virus (ISAV) from Cloned cDNA.

The piscine orthomyxovirus called infectious salmon anemia virus (ISAV) is one of the most important emerging pathogens affecting the salmon industry worldwide. The first reverse genetics system for ISAV, which allows the generation of recombinant ISA virus (rISAV), is an important tool for the characterization and study of this virus. The plasmid-based reverse genetics system for ISAV includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). The salmon, viral, and mammalian genetic elements included in the pSS-URG vectors allow the expression of the eight viral RNA segments. In addition to four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex, the eight pSS-URG vectors allowed the generation of infectious rISAV in salmon cells.

CX-5461 Preferentially Induces Top2α-Dependent DNA Breaks at Ribosomal DNA Loci.

While genotoxic chemotherapeutic agents are among the most effective tools to combat cancer, they are often associated with severe adverse effects caused by indiscriminate DNA damage in non-tumor tissue as well as increased risk of secondary carcinogenesis. This study builds on our previous work demonstrating that the RNA Polymerase I (Pol I) transcription inhibitor CX-5461 elicits a non-canonical DNA damage response and our discovery of a critical role for Topoisomerase 2α (Top2α) in the initiation of Pol I-dependent transcription. Here, we identify Top2α as a mediator of CX-5461 response in the murine Eµ-Myc B lymphoma model whereby sensitivity to CX-5461 is dependent on cellular Top2α expression/activity. Most strikingly, and in contrast to canonical Top2α poisons, we found that the Top2α-dependent DNA damage induced by CX-5461 is preferentially localized at the ribosomal DNA (rDNA) promoter region, thereby highlighting CX-5461 as a loci-specific DNA damaging agent. This mechanism underpins the efficacy of CX-5461 against certain types of cancer and can be used to develop effective non-genotoxic anticancer drugs.

The Wnt-dependent master regulator NKX1-2 controls mouse pre-implantation development.

Embryo size, specification, and homeostasis are regulated by a complex gene regulatory and signaling network. Here we used gene expression signatures of Wnt-activated mouse embryonic stem cell (mESC) clones to reverse engineer an mESC regulatory network. We identify NKX1-2 as a novel master regulator of preimplantation embryo development. We find that Nkx1-2 inhibition reduces nascent RNA synthesis, downregulates genes controlling ribosome biogenesis, RNA translation, and transport, and induces severe alteration of nucleolus structure, resulting in the exclusion of RNA polymerase I from nucleoli. In turn, NKX1-2 loss of function leads to chromosome missegregation in the 2- to 4-cell embryo stages, severe decrease in blastomere numbers, alterations of tight junctions (TJs), and impairment of microlumen coarsening. Overall, these changes impair the blastocoel expansion-collapse cycle and embryo cavitation, leading to altered lineage specification and developmental arrest.

The ribosomal protein L22 binds the MDM4 pre-mRNA and promotes exon skipping to activate p53 upon nucleolar stress.

The tumor suppressor p53 and its antagonists MDM2 and MDM4 integrate stress signaling. For instance, dysbalanced assembly of ribosomes in nucleoli induces p53. Here, we show that the ribosomal protein L22 (RPL22; eL22), under conditions of ribosomal and nucleolar stress, promotes the skipping of MDM4 exon 6. Upon L22 depletion, more full-length MDM4 is maintained, leading to diminished p53 activity and enhanced cellular proliferation. L22 binds to specific RNA elements within intron 6 of MDM4 that correspond to a stem-loop consensus, leading to exon 6 skipping. Targeted deletion of these intronic elements largely abolishes L22-mediated exon skipping and re-enables cell proliferation, despite nucleolar stress. L22 also governs alternative splicing of the L22L1 (RPL22L1) and UBAP2L mRNAs. Thus, L22 serves as a signaling intermediate that integrates different layers of gene expression. Defects in ribosome synthesis lead to specific alternative splicing, ultimately triggering p53-mediated transcription and arresting cell proliferation.

Identification of SL addition trans-splicing acceptor sites in the internal transcribed spacer I region of pre-rRNA in Leishmania (Leishmania) amazonensis

Trypanosomatidae is a family of early branching eukaryotes harbouring a distinctive repertoire of gene expression strategies. Functional mature messenger RNA is generated via the trans-splicing and polyadenylation processing of constitutively transcribed polycistronic units. Recently, trans-splicing of pre-small subunit ribosomal RNA in the 5' external transcribed spacer region and of precursor tRNAsec have been described. Here, we used a previously validated semi-nested reverse transcription-polymerase chain reaction strategy to investigate internal transcribed spacer (ITS) I acceptor sites in total RNA from Leishmania (Leishmania) amazonensis. Two distinct spliced leader-containing RNAs were detected indicating that trans-splicing reactions occur at two AG acceptor sites mapped in this ITS region. These data provide further evidence of the wide spectrum of RNA molecules that act as trans-splicing acceptors in trypanosomatids.

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.