RNA Polymerase II CTD phosphatase Rtr1 fine-tunes transcription termination.

RNA Polymerase II (RNAPII) transcription termination is regulated by the phosphorylation status of the C-terminal domain (CTD). The phosphatase Rtr1 has been shown to regulate serine 5 phosphorylation on the CTD; however, its role in the regulation of RNAPII termination has not been explored. As a consequence of RTR1 deletion, interactions within the termination machinery and between the termination machinery and RNAPII were altered as quantified by Disruption-Compensation (DisCo) network analysis. Of note, interactions between RNAPII and the cleavage factor IA (CF1A) subunit Pcf11 were reduced in rtr1Δ, whereas interactions with the CTD and RNA-binding termination factor Nrd1 were increased. Globally, rtr1Δ leads to decreases in numerous noncoding RNAs that are linked to the Nrd1, Nab3 and Sen1 (NNS) -dependent RNAPII termination pathway. Genome-wide analysis of RNAPII and Nrd1 occupancy suggests that loss of RTR1 leads to increased termination at noncoding genes. Additionally, premature RNAPII termination increases globally at protein-coding genes with a decrease in RNAPII occupancy occurring just after the peak of Nrd1 recruitment during early elongation. The effects of rtr1Δ on RNA expression levels were lost following deletion of the exosome subunit Rrp6, which works with the NNS complex to rapidly degrade a number of noncoding RNAs following termination. Overall, these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT cells to prevent premature termination of mRNAs and ncRNAs. Rtr1 facilitates low-level elongation of noncoding transcripts that impact RNAPII interference thereby shaping the transcriptome.

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation.

In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes.

Rrp6: Integrated roles in nuclear RNA metabolism and transcription termination.

The yeast RNA exosome is a eukaryotic ribonuclease complex essential for RNA processing, surveillance, and turnover. It is comprised of a barrel-shaped core and cap as well as a 3'-5' ribonuclease known as Dis3 that contains both endo- and exonuclease domains. A second exonuclease, Rrp6, is added in the nucleus. Dis3 and Rrp6 have both shared and distinct roles in RNA metabolism, and this review will focus primarily on Rrp6 and the roles of the RNA exosome in the nucleus. The functions of the nuclear exosome are modulated by cofactors and interacting partners specific to each type of substrate. Generally, the cofactor TRAMP (Trf4/5-Air2/1-Mtr4 polyadenylation) complex helps unwind unstable RNAs, RNAs requiring processing such as rRNAs, tRNAs, or snRNAs or improperly processed RNAs and direct it toward the exosome. In yeast, Rrp6 interacts with Nrd1, the cap-binding complex, and RNA polymerase II to aid in nascent RNA processing, termination, and polyA tail length regulation. Recent studies have shown that proper termination and processing of short, noncoding RNAs by Rrp6 is particularly important for transcription regulation across the genome and has important implications for regulation of diverse processes at the cellular level. Loss of proper Rrp6 and exosome activity may contribute to various pathologies such as autoimmune disease, neurological disorders, and cancer. WIREs RNA 2016, 7:91-104. doi: 10.1002/wrna.1317 For further resources related to this article, please visit the WIREs website.

The exosome component Rrp6 is required for RNA polymerase II termination at specific targets of the Nrd1-Nab3 pathway.

The exosome and its nuclear specific subunit Rrp6 form a 3'-5' exonuclease complex that regulates diverse aspects of RNA biology including 3' end processing and degradation of a variety of noncoding RNAs (ncRNAs) and unstable transcripts. Known targets of the nuclear exosome include short (<1000 bp) RNAPII transcripts such as small noncoding RNAs (snRNAs), cryptic unstable transcripts (CUTs), and some stable unannotated transcripts (SUTs) that are terminated by an Nrd1, Nab3, and Sen1 (NNS) dependent mechanism. NNS-dependent termination is coupled to RNA 3' end processing and/or degradation by the Rrp6/exosome in yeast. Recent work suggests Nrd1 is necessary for transcriptome surveillance, regulating promoter directionality and suppressing antisense transcription independently of, or prior to, Rrp6 activity. It remains unclear whether Rrp6 is directly involved in termination; however, Rrp6 has been implicated in the 3' end processing and degradation of ncRNA transcripts including CUTs. To determine the role of Rrp6 in NNS termination globally, we performed RNA sequencing (RNA-Seq) on total RNA and perform ChIP-exo analysis of RNA Polymerase II (RNAPII) localization. Deletion of RRP6 promotes hyper-elongation of multiple NNS-dependent transcripts resulting from both improperly processed 3' RNA ends and faulty transcript termination at specific target genes. The defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of depleting Nrd1 from the nucleus. Elongated transcripts were identified within all classes of known NNS targets with the largest changes in transcription termination occurring at CUTs. Interestingly, the extended transcripts that we have detected in our studies show remarkable similarity to Nrd1-unterminated transcripts at many locations, suggesting that Rrp6 acts with the NNS complex globally to promote transcription termination in addition to 3' end RNA processing and/or degradation at specific targets.

Phenotypic plasticity in normal breast derived epithelial cells.

BACKGROUND: Normal, healthy human breast tissue from a variety of volunteer donors has become available for research thanks to the establishment of the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB). Multiple epithelial (K-HME) and stromal cells (K-HMS) were established from the donated tissue. Explant culture was utilized to isolate the cells from pieces of breast tissue. Selective media and trypsinization were employed to select either epithelial cells or stromal cells. The primary, non-transformed epithelial cells, the focus of this study, were characterized by immunohistochemistry, flow cytometry, and in vitro cell culture. RESULTS: All of the primary, non-transformed epithelial cells tested have the ability to differentiate in vitro into a variety of cell types when plated in or on biologic matrices. Cells identified include stratified squamous epithelial, osteoclasts, chondrocytes, adipocytes, neural progenitors/neurons, immature muscle and melanocytes. The cells also express markers of embryonic stem cells. CONCLUSIONS: The cell culture conditions employed select an epithelial cell that is pluri/multipotent. The plasticity of the epithelial cells developed mimics that seen in metaplastic carcinoma of the breast (MCB), a subtype of triple negative breast cancer; and may provide clues to the origin of this particularly aggressive type of breast cancer. The KTB is a unique biorepository, and the normal breast epithelial cells isolated from donated tissue have significant potential as new research tools.

The interactome of the atypical phosphatase Rtr1 in Saccharomyces cerevisiae.

The phosphatase Rtr1 has been implicated in dephosphorylation of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) during transcription elongation and in regulation of nuclear import of RNAPII. Although it has been shown that Rtr1 interacts with RNAPII in yeast and humans, the specific mechanisms that underlie Rtr1 recruitment to RNAPII have not been elucidated. To address this, we have performed an in-depth proteomic analysis of Rtr1 interacting proteins in yeast. Our studies revealed that hyperphosphorylated RNAPII is the primary interacting partner for Rtr1. To extend these findings, we performed quantitative proteomic analyses of Rtr1 interactions in yeast strains deleted for CTK1, the gene encoding the catalytic subunit of the CTD kinase I (CTDK-I) complex. Interestingly, we found that the interaction between Rtr1 and RNAPII is decreased in ctk1Δ strains. We hypothesize that serine-2 CTD phosphorylation is required for Rtr1 recruitment to RNAPII during transcription elongation.

Lateral drill holes decrease strength of the femur: an observational study using finite element and experimental analyses.

BACKGROUND: Internal fixation of femoral fractures requires drilling holes through the cortical bone of the shaft of the femur. Intramedullary suction reduces the fat emboli produced by reaming and nailing femoral fractures but requires four suction portals to be drilled into the femoral shaft. This work investigated the effect of these additional holes on the strength of the femur. METHODS: Finite element analysis (FEA) was used to calculate compression, tension and load limits which were then compared to the results from mechanical testing. Models of intact femora and fractured femora internally fixed with intramedullary nailing were generated. In addition, four suction portals, lateral, anterior and posterior, were modelled. Stresses were used to calculate safety factors and predict fatigue. Physical testing on synthetic femora was carried out on a universal mechanical testing machine. RESULTS: The FEA model for stresses generated during walking showed tensile stresses in the lateral femur and compression stresses in the medial femur with a maximum sheer stress through the neck of the femur. The lateral suction portals produced tensile stresses up to over 300% greater than in the femur without suction portals. The anterior and posterior portals did not significantly increase stresses. The lateral suction portals had a safety factor of 0.7, while the anterior and posterior posts had safety factors of 2.4 times walking loads. Synthetic bone subjected to cyclical loading and load to failure showed similar results. On mechanical testing, all constructs failed at the neck of the femur. CONCLUSIONS: The anterior suction portals produced minimal increases in stress to loading so are the preferred site should a femur require such drill holes for suction or internal fixation.

Medical genetics and epigenetics of telomerase.

Telomerase is a specialized reverse transcriptase that extends and maintains the terminal ends of chromosomes, or telomeres. Since its discovery in 1985 by Nobel Laureates Elizabeth Blackburn and Carol Greider, thousands of articles have emerged detailing its significance in telomere function and cell survival. This review provides a current assessment on the importance of telomerase regulation and relates it in terms of medical genetics. In this review, we discuss the recent findings on telomerase regulation, focusing on epigenetics and non-coding RNAs regulation of telomerase, such as microRNAs and the recently discovered telomeric-repeat containing RNA transcripts. Human genetic disorders that develop due to mutations in telomerase subunits, the role of single nucleotide polymorphisms in genes encoding telomerase components and diseases as a result of telomerase regulation going awry are also discussed. Continual investigation of the complex regulation of telomerase will further our insight into the use of controlling telomerase activity in medicine.

The telomerase template antagonist GRN163L alters MDA-MB-231 breast cancer cell morphology, inhibits growth, and augments the effects of paclitaxel.

Telomeres are repetitive (TTAGGG)(n) DNA sequences found at the end of chromosomes that protect the ends from recombination, end to end fusions, and recognition as damaged DNA. Telomerase activity can be detected in 85% to 90% of human tumors, which stabilizes telomeres to prevent apoptosis or cellular senescence. Previous reports showed the efficacy of the novel telomerase template antagonist, GRN163L, as a potential anticancer agent. The objective of the present study was to elucidate the molecular effects of GRN163L in MDA-MB-231 breast cancer cells and to determine whether GRN163L could be used in mechanism-based combination therapy for breast cancer. We observed that GRN163L reduced MDA-MB-231 growth rates without a significant effect on breast cancer cell viability within the first 14 days in vitro. In addition, GRN163L altered cell morphology, actin filament organization, and focal adhesion formation in MDA-MB-231 cells. Importantly, the cellular response to GRN163L significantly augmented the effects of the microtubule stabilizer paclitaxel in MDA-MB-231 breast cancer cell growth in vitro and in vivo compared with paclitaxel alone or a mismatch control oligonucleotide plus paclitaxel. Furthermore, in vitro MDA-MB-231 invasive potential was significantly inhibited with GRN163L and paclitaxel. These data support a rationale for potentially combining GRN163L with paclitaxel for the treatment of breast cancer in the clinical setting.