Vaccination of cattle with a virus vector vaccine against a major membrane protein of Mycobacterium avium subsp. paratuberculosis elicits CD8 cytotoxic T cells that kill intracellular bacteria.

Analysis of the recall response ex vivo in cattle vaccinated with a Mycobacterium avium subsp. paratuberculosis (Map) rel deletion mutant revealed the immune response was directed toward a 35 kD major membrane protein (MMP) of Map. Antigen presenting cells (APC) primed with MMP elicited expansion of CD8 cytotoxic memory T cells (CTL) with ability to kill intracellular bacteria. Development of CTL was MHC-restricted. The gene MAP2121c, encoding MMP, was modified for expression of MMP (tPA-MMP-2mut) in a mammalian cell line to explore the potential of developing MMP as a vaccine. Ex vivo stimulation of PBMC, from Map free cattle, with APC primed with tPA-MMP-2mut expressed p35 elicited a primary CD8 CTL response comparable to the recall response elicited with PBMC from cattle vaccinated with either the Maprel deletion mutant or MMP. In the present study, the modified gene for MMP, now referred to as p35NN, was placed into a bovine herpes virus-4 (BoHV4) vector to determine the potential use of BoHV-4AΔTK-p35NN as a peptide-based vaccine. Subcutaneous vaccination of healthy cattle with BoHV-4AΔTK-p35NN elicited a CTL recall response, as detected ex vivo. The results show use of a virus vector is an effective way for delivery of MMP as a vaccine. The immunogenic activity of MMP was not lost when modified for expression in mammalian cells. The next step is to conduct a field trial to determine if presence of an immune response to MMP prevents Map from establishing an infection.

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.

Validation of a real-time quaking-induced conversion (RT-QuIC) assay protocol to detect chronic wasting disease using rectal mucosa of naturally infected, pre-clinical white-tailed deer (Odocoileus virginianus).

Chronic wasting disease (CWD) is a fatal prion disease of cervids spreading across North America. More effective mitigation efforts may require expansion of the available toolkit to include new methods that provide earlier antemortem detection, higher throughput, and less expense than current immunohistochemistry (IHC) methods. The rectal mucosa near the rectoanal junction is a site of early accumulation of CWD prions and is safely sampled in living animals by pinch biopsy. A fluorescence-based, 96-well format, protein-aggregation assay-the real-time quaking-induced conversion (RT-QuIC) assay-is capable of ultra-sensitive detection of CWD prions. Notably, the recombinant protein substrate is crucial to the assay's performance and is now commercially available. In this blinded independent study, the preclinical diagnostic performance of a standardized RT-QuIC protocol using a commercially sourced substrate (MNPROtein) and a laboratory-produced substrate was studied using mock biopsy samples of the rectal mucosa from 284 white-tailed deer (Odocoileus virginianus). The samples were from a frozen archive of intact rectoanal junctions collected at depopulations of farmed herds positive for CWD in the United States. All deer were pre-clinical at the time of depopulation and infection status was established from the regulatory record, which evaluated the medial retropharyngeal lymph nodes (MRPLNs) and obex by CWD-IHC. A pre-analytic sample precipitation step was found to enhance the protocol's detection limit. Performance metrics were influenced by the choice of RT-QuIC diagnostic cut points (minimum number of positive wells and assay time) and by deer attributes (preclinical infection stage and prion protein genotype). The peak overall diagnostic sensitivities of the protocol were similar for both substrates (MNPROtein, 76.8%; laboratory-produced, 73.2%), though each was achieved at different cut points. Preclinical infection stage and prion protein genotype at codon 96 (G = glycine, S = serine) were primary predictors of sensitivity. The diagnostic sensitivities in late preclinical infections (CWD-IHC positive MPRLNs and obex) were similar, ranging from 96% in GG96 deer to 80% in xS96 deer (x = G or S). In early preclinical infections (CWD-IHC positive MRPLNs only), the diagnostic sensitivity was 64-71% in GG96 deer but only 25% in xS96 deer. These results demonstrate that this standardized RT-QuIC protocol for rectal biopsy samples using a commercial source of substrate produced stratified diagnostic sensitivities similar to or greater than those reported for CWD-IHC but in less than 30 hours of assay time and in a 96-well format. Notably, the RT-QuIC protocol used herein represents a standardization of protocols from several previous studies. Alignment of the sensitivities across these studies suggests the diagnostic performance of the assay is robust given quality reagents, optimized diagnostic criteria, and experienced staff.

Reversible Kinetics in Multi-nucleotide Addition Catalyzed by S. cerevisiae RNA polymerase II Reveal Slow Pyrophosphate Release.

Eukaryotes express at least three nuclear DNA dependent RNA polymerases (Pols). Pols I, II, and III synthesize ribosomal (r) RNA, messenger (m) RNA, and transfer (t) RNA, respectively. Pol I and Pol III have intrinsic nuclease activity conferred by the A12.2 and C11 subunits, respectively. In contrast, Pol II requires the transcription factor (TF) IIS to confer robust nuclease activity. We recently reported that in the absence of the A12.2 subunit Pol I reverses bond formation by pyrophosphorolysis in the absence of added PPi, indicating slow PPi release. Thus, we hypothesized that Pol II, naturally lacking TFIIS, would reverse bond formation through pyrophosphorolysis. Here we report the results of transient-state kinetic experiments to examine the addition of nine nucleotides to a growing RNA chain catalyzed by Pol II. Our results indicate that Pol II reverses bond formation by pyrophosphorolysis in the absence of added PPi. We propose that, in the absence of endonuclease activity, this bond reversal may represent kinetic proofreading. Thus, given the hypothesis that Pol I evolved from Pol II through the incorporation of general transcription factors, pyrophosphorolysis may represent a more ancient form of proofreading that has been evolutionarily replaced with nuclease activity.

Sensitive detection of pathological seeds of α-synuclein, tau and prion protein on solid surfaces.

Prions or prion-like aggregates such as those composed of PrP, α-synuclein, and tau are key features of proteinopathies such as prion, Parkinson's and Alzheimer's diseases, respectively. Their presence on solid surfaces may be biohazardous under some circumstances. PrP prions bound to solids are detectable by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays if the solids can be immersed in assay wells or the prions transferred to pads. Here we show that prion-like seeds can remain detectable on steel wires for at least a year, or even after enzymatic cleaning and sterilization. We also show that contamination of larger objects with pathological seeds of α-synuclein, tau, and PrP can be detected by simply assaying a sampling medium that has been transiently applied to the surface. Human α-synuclein seeds in dementia with Lewy bodies brain tissue were detected by α-synuclein RT-QuIC after drying of tissue dilutions with concentrations as low as 10-6 onto stainless steel. Tau RT-QuIC detected tau seeding activity on steel exposed to Alzheimer's disease brain tissue diluted as much as a billion fold. Prion RT-QuIC assays detected seeding activity on plates exposed to brain dilutions as extreme as 10-5-10-8 from prion-affected humans, sheep, cattle and cervids. Sampling medium collected from surgical instruments used in necropsies of sporadic Creutzfeldt-Jakob disease-infected transgenic mice was positive down to 10-6 dilution. Sensitivity for prion detection was not sacrificed by omitting the recombinant PrP substrate from the sampling medium during its application to a surface and subsequent storage as long as the substrate was added prior to performing the assay reaction. Our findings demonstrate practical prototypic surface RT-QuIC protocols for the highly sensitive detection of pathologic seeds of α-synuclein, tau, and PrP on solid objects.

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.

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.

Quantifying the impact of initial RNA primer length on nucleotide addition by RNA polymerase I.

Transient state kinetic studies of eukaryotic DNA-dependent RNA polymerases (Pols) in vitro provide quantitative characterization of enzyme activity at the level of individual nucleotide addition events. Previous work revealed heterogeneity in the rate constants governing nucleotide addition by yeast RNA polymerase I (Pol I) for each position on a template DNA. In contrast, the rate constants that described nucleotide addition by yeast RNA polymerase II (Pol II) were more homogeneous. This observation led to the question, what drives the variability of rate constants governing RNA synthesis by Pol I? Are the kinetics of nucleotide addition dictated by the position of the nascent RNA within the polymerase or by the identity of the next encoded nucleotide? In this study, we examine the impact of nucleotide position (i.e. nascent RNA primer length) on the rate constants governing nine sequential nucleotide addition events catalyzed by Pol I. The results reveal a conserved trend in the observed rate constants at each position for all primer lengths used, and highlight that the 9-nucleotide, or 9-mer, RNA primer provides the fastest observed rate constants. These findings suggest that the observed heterogeneity of rate constants for RNA synthesis by Pol I in vitro is driven primarily by the template sequence.

B-cell leukemia in an adult sheep.

B-cell leukemia is a rare form of hematologic neoplasia in sheep, especially in adult animals. We present a case report of a 5-year-old WhiteFace Sheep wether with suspected acute lymphoblastic leukemia. The patient, a second-generation relative of ewes experimentally inoculated with atypical scrapie, exhibited acute lethargy and loss of appetite. Laboratory investigation revealed marked leukocytosis, lymphocytosis, and abnormal serum chemistry panel results. Microscopic examination of blood and bone marrow smears exhibited a high percentage of large neoplastic cells with lymphoid characteristics. Histopathologic analysis of the spleen, liver, lungs, and other organs confirmed the presence of widespread tissue infiltration by neoplastic cells. Immunohistochemical labeling demonstrated strong intracytoplasmic labeling for CD20, consistent with B-cell neoplasia. Flow cytometric analysis confirmed the B-cell lineage of the neoplastic cells. Screening for bovine leukemia virus, which can experimentally cause leukemia in sheep, yielded a negative result. In this case, the diagnosis of B-cell leukemia was supported by a comprehensive panel of diagnostic evaluations, including cytology, histopathology, immunohistochemistry, and immunophenotyping. This case report highlights the significance of accurate diagnosis and classification of hematologic neoplasia in sheep, emphasizing the need for immunophenotyping to aid in the diagnosis of B-cell leukemia. It also emphasizes the importance of considering spontaneous leukemia as a differential diagnosis in sheep with lymphoid neoplasia, especially in the absence of circulating infectious diseases.

Biphasic pleural mesothelioma in a goat.

An 8-year-old Saanen goat doe was seen for inappetence, tachycardia, and intermittent bluish-grey discoloration of the oral mucous membranes. On physical examination, the goat was mildly tachypneic and tachycardic, with reduced sounds auscultated on the left side of the thorax. Euthanasia was elected. Necropsy revealed an infiltrative, multinodular mass within the left thoracic cavity and innumerable small, tan nodules disseminated across the pleura of the lungs, thoracic walls, and diaphragm. Upon histologic examination, the mass was composed of highly pleomorphic, fusiform to polygonal cells. Neoplastic cells exhibited positive immunoreactivity for both cytokeratin and vimentin, consistent with a diagnosis of biphasic pleural mesothelioma. Key clinical message: Mesothelioma has rarely been described in the goat but should be considered as a differential diagnosis for thoracic masses in small ruminants, along with thymoma; metastatic neoplasia; carcinomatosis; and granulomatous lesions caused by parasites, bacteria, and fungi.

Mésothéliome pleural biphasique chez une chèvre. Une chèvre Saanen âgée de 8 ans a été vue pour de l'inappétence, une tachycardie et une décoloration gris bleuâtre intermittente des muqueuses buccales. À l'examen physique, la chèvre était légèrement tachypnéique et tachycardique, avec des sons réduits auscultés du côté gauche du thorax. Il a été décidé d'euthanasier l'animal. L'autopsie a révélé une masse multinodulaire infiltrante dans la cavité thoracique gauche et d'innombrables petits nodules brun clair disséminés à travers la plèvre pulmonaire, les parois thoraciques et le diaphragme. À l'examen histologique, la masse était composée de cellules hautement pléomorphes, fusiformes à polygonales. Les cellules néoplasiques ont présenté une immunoréactivité positive pour la cytokératine et la vimentine, compatible avec un diagnostic de mésothéliome pleural biphasique.Message clinique clé:Le mésothéliome a rarement été décrit chez la chèvre mais doit être considéré comme un diagnostic différentiel des masses thoraciques chez les petits ruminants, avec le thymome, la néoplasie métastatique, la carcinomatose et les lésions granulomateuses causées par des parasites, des bactéries et des champignons.(Traduit par Dr Serge Messier).

Small Molecule RBI2 Disrupts Ribosome Biogenesis through Pre-rRNA Depletion.

Cancer cells are especially sensitive to perturbations in ribosome biogenesis as they rely on finely tuned protein homeostasis to facilitate their rapid growth and proliferation. While ribosome synthesis and cancer have a well-established relationship, ribosome biogenesis has only recently drawn interest as a cancer therapeutic target. In this study, we exploited the relationship between ribosome biogenesis and cancer cell proliferation by using a potent ribosome biogenesis inhibitor, RBI2 (Ribosome Biogenesis Inhibitor 2), to perturb cancer cell growth and viability. We demonstrate herein that RBI2 significantly decreases cell viability in malignant melanoma cells and breast cancer cell lines. Treatment with RBI2 dramatically and rapidly decreased ribosomal RNA (rRNA) synthesis, without affecting the occupancy of RNA polymerase I (Pol I) on the ribosomal DNA template. Next-generation RNA sequencing (RNA-seq) revealed that RBI2 and previously described ribosome biogenesis inhibitor CX-5461 induce distinct changes in the transcriptome. An investigation of the content of the pre-rRNAs through RT-qPCR revealed an increase in the polyadenylation of cellular rRNA after treatment with RBI2, constituting a known pathway by which rRNA degradation occurs. Northern blotting revealed that RBI2 does not appear to impair or alter rRNA processing. Collectively, these data suggest that RBI2 inhibits rRNA synthesis differently from other previously described ribosome biogenesis inhibitors, potentially acting through a novel pathway that upregulates the turnover of premature rRNAs.

The A12.2 Subunit Plays an Integral Role in Pyrophosphate Release of RNA Polymerase I.

RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA), which is the first and rate-limiting step in ribosome biosynthesis. A12.2 (A12) is a critical subunit of Pol I that is responsible for activating Pol I's exonuclease activity. We previously reported a kinetic mechanism for single-nucleotide incorporation catalyzed by Pol I lacking the A12 subunit (ΔA12 Pol I) purified from S. cerevisae and revealed that ΔA12 Pol I exhibited much slower incorporation compared to Pol I. However, it is unknown if A12 influences each nucleotide incorporation in the context of transcription elongation. Here, we show that A12 contributes to every repeating cycle of nucleotide addition and that deletion of A12 results in an entirely different kinetic mechanism compared to WT Pol I. We found that instead of one irreversible step between each nucleotide addition cycle, as reported for wild type (WT) Pol I, the ΔA12 variant requires one reversible step to describe each nucleotide addition. Reversibility fundamentally requires slow PPi release. Consistently, we show that Pol I is more pyrophosphate (PPi) concentration dependent than ΔA12 Pol I. This observation supports the model that PPi is retained in the active site of ΔA12 Pol I longer than WT Pol I. These results suggest that A12 promotes PPi release, revealing a larger role for the A12.2 subunit in the nucleotide addition cycle beyond merely activating exonuclease activity.

Expression of RNA polymerase I catalytic core is influenced by RPA12.

RNA Polymerase I (Pol I) has recently been recognized as a cancer therapeutic target. The activity of this enzyme is essential for ribosome biogenesis and is universally activated in cancers. The enzymatic activity of this multi-subunit complex resides in its catalytic core composed of RPA194, RPA135, and RPA12, a subunit with functions in RNA cleavage, transcription initiation and elongation. Here we explore whether RPA12 influences the regulation of RPA194 in human cancer cells. We use a specific small-molecule Pol I inhibitor BMH-21 that inhibits transcription initiation, elongation and ultimately activates the degradation of Pol I catalytic subunit RPA194. We show that silencing RPA12 causes alterations in the expression and localization of Pol I subunits RPA194 and RPA135. Furthermore, we find that despite these alterations not only does the Pol I core complex between RPA194 and RPA135 remain intact upon RPA12 knockdown, but the transcription of Pol I and its engagement with chromatin remain unaffected. The BMH-21-mediated degradation of RPA194 was independent of RPA12 suggesting that RPA12 affects the basal expression, but not the drug-inducible turnover of RPA194. These studies add to knowledge defining regulatory factors for the expression of this Pol I catalytic subunit.

Protocol for monitoring and analyzing single nucleotide incorporation by S. cerevisiae RNA polymerases.

Here we present an optimized protocol for monitoring and analyzing single nucleotide incorporation by RNA polymerases. This protocol describes the assembly of Saccharomyces cerevisiae RNA polymerase I elongation complexes in a promoter-independent system in vitro. We describe how to collect a time course using a quench-flow, a rapid mixing instrument, and subsequently resolve reactions on a polyacrylamide gel. Finally, we detail how to quantify the gel images. For complete details on the use and execution of this protocol, please refer to Appling et al. (2015).1.

Tonsil biopsy to detect chronic wasting disease in white-tailed deer (Odocoileus virginianus) by immunohistochemistry.

Chronic wasting disease (CWD) continues to spread in wild and farmed cervid populations. Early antemortem CWD testing of farmed cervids is of considerable interest to producers and regulatory agencies as a tool to combat this spread. The tissues accessible for antemortem sampling are limited and include biopsy of the tonsil and recto-anal mucosa-associated lymphoid tissue (RAMALT). The sensitivity to detect CWD by immunohistochemistry (IHC)-the regulatory gold standard-using biopsy samples of RAMALT from naturally infected white-tailed deer (WTD) has been determined by several studies. However, similar information is lacking for tonsil biopsy. In this study, two-bite tonsil biopsies from 79 naturally infected farmed WTD were used to determine the diagnostic sensitivity of tonsil IHC compared to the official CWD status based on results from the medial retropharyngeal lymph nodes and obex. IHC detection of CWD by tonsil biopsy was compared to the result and follicle metrics from the contralateral whole tonsil. The sensitivity of two-bite tonsil biopsy for detecting CWD by IHC was 72% overall. When the stage of infection was considered, the sensitivity was 92% for deer in late preclinical infection but only 55% for early preclinical infection. For deer with early preclinical infection, the sensitivity for deer homozygous for the prion protein gene (PRNP) coding for glycine at codon 96 (GG) was 66% but only 30% when heterozygous for the serine substitution (GS). The results indicate that the sensitivity of two-bite tonsil biopsy in WTD, and consequently its potential utility as an antemortem diagnostic, is limited during early infection, especially in WTD heterozygous for the serine substitution at PRNP codon 96.

Transient-State Kinetic Analysis of the RNA Polymerase II Nucleotide Incorporation Mechanism.

Eukaryotic RNA polymerase II (Pol II) is an essential enzyme that lies at the core of eukaryotic biology. Due to its pivotal role in gene expression, Pol II has been subjected to a substantial number of investigations. We aim to further our understanding of Pol II nucleotide incorporation by utilizing transient-state kinetic techniques to examine Pol II single nucleotide addition on the millisecond time scale. We analyzed Saccharomyces cerevisiae Pol II incorporation of ATP or an ATP analog, Sp-ATP-α-S. Here we have measured the rate constants governing individual steps of the Pol II transcription cycle in the presence of ATP or Sp-ATP-α-S. These results suggest that Pol II catalyzes nucleotide incorporation by binding the next cognate nucleotide and immediately catalyzes bond formation and bond formation is either followed by a conformational change or pyrophosphate release. By comparing our previously published RNA polymerase I (Pol I) and Pol I lacking the A12 subunit (Pol I ΔA12) results that we collected under the same conditions with the identical technique, we show that Pol II and Pol I ΔA12 exhibit similar nucleotide addition mechanisms. This observation indicates that removal of the A12 subunit from Pol I results in a Pol II like enzyme. Taken together, these data further our collective understanding of Pol II's nucleotide incorporation mechanism and the evolutionary divergence of RNA polymerases across the three domains of life.

RNA Polymerase I Is Uniquely Vulnerable to the Small-Molecule Inhibitor BMH-21.

Cancer cells require robust ribosome biogenesis to maintain rapid cell growth during tumorigenesis. Because RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and rate-limiting step of ribosome biogenesis, it has emerged as a promising anti-cancer target. Over the last decade, novel cancer therapeutics targeting Pol I have progressed to clinical trials. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and represses cancer cell growth. Several recent studies have uncovered key mechanisms by which BMH-21 inhibits ribosome biosynthesis but the selectivity of BMH-21 for Pol I has not been directly measured. Here, we quantify the effects of BMH-21 on Pol I, RNA polymerase II (Pol II), and RNA polymerase III (Pol III) in vitro using purified components. We found that BMH-21 directly impairs nucleotide addition by Pol I, with no or modest effect on Pols II and III, respectively. Additionally, we found that BMH-21 does not affect the stability of any of the Pols' elongation complexes. These data demonstrate that BMH-21 directly exploits unique vulnerabilities of Pol I.

Identification of an E3 ligase that targets the catalytic subunit of RNA Polymerase I upon transcription stress.

RNA Polymerase I (Pol I) synthesizes rRNA, which is the first and rate-limiting step in ribosome biogenesis. Factors governing the stability of the polymerase complex are not known. Previous studies characterizing Pol I inhibitor BMH-21 revealed a transcriptional stress-dependent pathway for degradation of the largest subunit of Pol I, RPA194. To identify the E3 ligase(s) involved, we conducted a cell-based RNAi screen for ubiquitin pathway genes. We establish Skp-Cullin-F-box protein complex F-box protein FBXL14 as an E3 ligase for RPA194. We show that FBXL14 binds to RPA194 and mediates RPA194 ubiquitination and degradation in cancer cells treated with BMH-21. Mutation analysis in yeast identified lysines 1150, 1153, and 1156 on Rpa190 relevant for the protein degradation. These results reveal the regulated turnover of Pol I, showing that the stability of the catalytic subunit is controlled by the F-box protein FBXL14 in response to transcription stress.

Rate of transcription elongation and sequence-specific pausing by RNA polymerase I directly influence rRNA processing.

One of the first steps in ribosome biogenesis is transcription of the ribosomal DNA by RNA polymerase I (Pol I). Processing of the resultant rRNA begins cotranscriptionally, and perturbation of Pol I transcription elongation results in defective rRNA processing. Mechanistic insight regarding the link between transcription elongation and ribosome assembly is lacking because of limited in vivo methods to assay Pol I transcription. Here, we use native elongating transcript sequencing (NET-Seq) with a strain of Saccharomyces cerevisiae containing a point mutation in Pol I, rpa190-F1205H, which results in impaired rRNA processing and ribosome assembly. We previously demonstrated that this mutation caused a mild reduction in the transcription elongation rate of Pol I in vitro; however, transcription elongation by the mutant has not been characterized in vivo. Here, our findings demonstrate that the mutant Pol I has an increased pause propensity during processive transcription elongation both in vitro and in vivo. NET-Seq reveals that rpa190-F1205H Pol I displays alternative pause site preferences in vivo. Specifically, the mutant is sensitized to A/G residues in the RNA:DNA hybrid and at the last incorporated nucleotide position. Furthermore, both NET-Seq and EM analysis of Miller chromatin spreads reveal pileups of rpa190-F1205H Pol I throughout the ribosomal DNA, particularly at the 5' end of the 35S gene. This combination of in vitro and in vivo analyses of a Pol I mutant provides novel insights into Pol I elongation properties and indicates how these properties are crucial for efficient cotranscriptional rRNA processing and ribosome assembly.

Uncovering the mechanisms of transcription elongation by eukaryotic RNA polymerases I, II, and III.

Eukaryotes express three nuclear RNA polymerases (Pols I, II, and III) that are essential for cell survival. Despite extensive investigation of the three Pols, significant knowledge gaps regarding their biochemical properties remain because each Pol has been evaluated independently under disparate experimental conditions and methodologies. To advance our understanding of the Pols, we employed identical in vitro transcription assays for direct comparison of their elongation rates, elongation complex (EC) stabilities, and fidelities. Pol I is the fastest, most likely to misincorporate, forms the least stable EC, and is most sensitive to alterations in reaction buffers. Pol II is the slowest of the Pols, forms the most stable EC, and negligibly misincorporated an incorrect nucleotide. The enzymatic properties of Pol III were intermediate between Pols I and II in all assays examined. These results reveal unique enzymatic characteristics of the Pols that provide new insights into their evolutionary divergence.