Proteomic Characterization of Cerebrospinal Fluid from Ataxia-Telangiectasia (A-T) Patients Using a LC/MS-Based Label-Free Protein Quantification Technology

Cerebrospinal fluid (CSF) has been used for biomarker discovery of neurodegenerative diseases in humans since biological changes in the brain can be seen in this biofluid. Inactivation of A-T-mutated protein (ATM), a multifunctional protein kinase, is responsible for A-T, yet biochemical studies have not succeeded in conclusively identifying the molecular mechanism(s) underlying the neurodegeneration seen in A-T patients or the proteins that can be used as biomarkers for neurologic assessment of A-T or as potential therapeutic targets. In this study, we applied a high-throughput LC/MS-based label-free protein quantification technology to quantitatively characterize the proteins in CSF samples in order to identify differentially expressed proteins that can serve as potential biomarker candidates for A-T. Among 204 identified CSF proteins with high peptide-identification confidence, thirteen showed significant protein expression changes. Bioinformatic analysis revealed that these 13 proteins are either involved in neurodegenerative disorders or cancer. Future molecular and functional characterization of these proteins would provide more insights into the potential therapeutic targets for the treatment of A-T and the biomarkers that can be used to monitor or predict A-T disease progression. Clinical validation studies are required before any of these proteins can be developed into clinically useful biomarkers

UBF levels determine the number of active ribosomal RNA genes in mammals

In mammals, the mechanisms regulating the number of active copies of the ∼200 ribosomal RNA (rRNA) genes transcribed by RNA polymerase I are unclear. We demonstrate that depletion of the transcription factor upstream binding factor (UBF) leads to the stable and reversible methylation-independent silencing of rRNA genes by promoting histone H1–induced assembly of transcriptionally inactive chromatin. Chromatin remodeling is abrogated by the mutation of an extracellular signal-regulated kinase site within the high mobility group box 1 domain of UBF1, which is required for its ability to bend and loop DNA in vitro. Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased. We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression. Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation

Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription

Transcription of the ribosomal RNA genes (rDNA) by RNA polymerase I (Pol I) is a major control step for ribosome synthesis and is tightly linked to cellular growth. However, the question of whether this process is modulated primarily at the level of transcription initiation or elongation is controversial. Studies in markedly different cell types have identified either initiation or elongation as the major control point. In this study, we have re-examined this question in NIH3T3 fibroblasts using a combination of metabolic labeling of the 47S rRNA, chromatin immunoprecipitation analysis of Pol I and overexpression of the transcription initiation factor Rrn3. Acute manipulation of growth factor levels altered rRNA synthesis rates over 8-fold without changing Pol I loading onto the rDNA. In fact, robust changes in Pol I loading were only observed under conditions where inhibition of rDNA transcription was associated with chronic serum starvation or cell cycle arrest. Overexpression of the transcription initiation factor Rrn3 increased loading of Pol I on the rDNA but failed to enhance rRNA synthesis in either serum starved, serum treated or G0/G1 arrested cells. Together these data suggest that transcription elongation is rate limiting for rRNA synthesis. We propose that transcription initiation is required for rDNA transcription in response to cell cycle cues, whereas elongation controls the dynamic range of rRNA synthesis output in response to acute growth factor modulation

Characterization of the Interactions of Mammalian RNA Polymerase I Associated Proteins PAF53 and PAF49

Masami Muramatsu’s laboratory demonstrated the critical role of RNA polymerase I (Pol I)-associated factor PAF53 in mammalian rRNA transcription. They have also identified a second polymerase associated factor, PAF49. Both PAF49 and PAF53 copurify with that fraction of the RNA polymerase I molecules that can function in transcription initiation <i>in vitro</i>. PAF49 and PAF53 are the mammalian homologues of two subunits of yeast RNA polymerase I, A34.5 and A49, that form a TFIIF-related subcomplex in yeast RNA polymerase I. In light of those publications, we investigated the interactions between various deletion and substitution mutants of mammalian PAF49 and PAF53 with the purpose of identifying those domains of the mammalian proteins that interact. Comparison of our results with structural studies on yeast A34.5 and A49 demonstrates that the yeast and mammalian proteins may in fact share structural similarities. In fact, the deletion mutagenesis data confirmed and extended the structural studies. For example, amino acids 41–86 of PAF49 were sufficient to provide the basis for heterodimerization. <i>In silico</i> structural analysis predicted that this region could assume a structure similar to the homologous region of yeast A34.5. Those similarities are insufficient, by themselves, for the proteins to form interspecific heterodimers. However, substitution of amino acids 52–98 of yeast A34.5 with amino acids 41–86 of mammalian PAF49 resulted in a protein that could heterodimerize with mouse PAF53

Presence of elongation factor 1 in nuclei and nucleoli of rat liver

Purified rat liver nuclei contain 11% of the total cellular translation elongation factor 1 activity. Ninety percent of the nuclear EF-1 activity was in the nucleoplasm and 10% was nucleolar. The specific activities of the nuclear and nucleolar EF-1 were 2 to 3 times higher, respectively, than EF-1 activity of the liver homogenate. The presence of EF-1 in the purified nuclei did not result from cytoplasmic contamination since only 0.14% of the cellular lactate dehydrogenase was present in the nuclei. These results provide the first evidence for the presence in the cell nucleus of translational factors of protein synthesis

Phosphorylation of Eukaryotic Translation Initiation Factor 2α Coordinates rRNA Transcription and Translation Inhibition during Endoplasmic Reticulum Stress▿ †

The endoplasmic reticulum (ER) is the major cellular compartment where folding and maturation of secretory and membrane proteins take place. When protein folding needs exceed the capacity of the ER, the unfolded protein response (UPR) pathway modulates gene expression and downregulates protein translation to restore homeostasis. Here, we report that the UPR downregulates the synthesis of rRNA by inactivation of the RNA polymerase I basal transcription factor RRN3/TIF-IA. Inhibition of rRNA synthesis does not appear to involve the well-characterized mTOR (mammalian target of rapamycin) pathway; instead, PERK-dependent phosphorylation of eIF2α plays a critical role in the inactivation of RRN3/TIF-IA. Downregulation of rRNA transcription occurs simultaneously or slightly prior to eIF2α phosphorylation-induced translation repression. Since rRNA is the most abundant RNA species, constituting ∼90% of total cellular RNA, its downregulation exerts a significant impact on cell physiology. Our study demonstrates the first link between regulation of translation and rRNA synthesis with phosphorylation of eIF2α, suggesting that this pathway may be broadly utilized by stresses that activate eIF2α kinases in order to coordinately regulate translation and ribosome biogenesis during cellular stress

The Three-dimensional Study of Chromosomes and Upstream Binding Factor-immunolabeled Nucleolar Organizer Regions Demonstrates Their Nonrandom Spatial Arrangement during Mitosis

The volumic rearrangement of both chromosomes and immunolabeled upstream binding factor in entire well-preserved mitotic cells was studied by confocal microscopy. By using high-quality three-dimensional visualization and tomography, it was possible to investigate interactively the volumic organization of chromosome sets and to focus on their internal characteristics. More particularly, this study demonstrates the nonrandom positioning of metaphase chromosomes bearing nucleolar organizer regions as revealed by their positive upstream binding factor immunolabeling. During the complex morphogenesis of the progeny nuclei from anaphase to late telophase, the equal partitioning of the nucleolar organizer regions is demonstrated by quantification, and their typical nonrandom central positioning within the chromosome sets is revealed

RNA polymerase I transcription in confluent cells: Rb downregulates rDNA transcription during confluence-induced cell cycle arrest

When 3T6 cells are confluent, they withdraw from the cell cycle. Concomitant with cell cycle arrest a significant reduction in RNA polymerase I transcription (80% decrease at 100% confluence) is observed. In the present study, we examined mechanism(s) through which transcription of the ribosomal genes is coupled to cell cycle arrest induced by cell density. Interestingly with an increase in cell density (from 3–43% confluence), a significant accumulation in the cellular content of hyperphosphorylated Rb was observed. As cell density increased further, the hypophosphorylated form of Rb became predominant and accumulated in the nucleoli. Co-immunoprecipitation experiments demonstrated there was also a significant rise in the amount of hypophosphorylated Rb associated with the rDNA transcription factor UBF. This increased interaction between Rb and UBF correlated with the reduced rate of rDNA transcription. Furthermore, overexpression of recombinant Rb inhibited UBF-dependent activation of transcription from a cotransfected rDNA reporter in either confluent or exponential cells. The amounts or activities of the rDNA transcription components we examined did not significantly change with cell cycle arrest. Although the content of PAF53, a polymerase associated factor, was altered marginally (decreased 38%), the time course and magnitude of the decrease did not correlate with the reduced rate of rDNA transcription. The results presented support a model wherein regulation of the binding of UBF to Rb and, perhaps the cellular content of PAF53, are components of the mechanism through which cell cycle and rDNA transcription are linked.This study was supported in part by National Institute of Health grants GM48991 (LI Rothblum), DK15658 (LS Jefferson) and DK13499 (LS Jefferson), the Juvenile Diabetes Foundation JDFI195051 (LS Jefferson) and an award from the Geisinger Foundation (LI Rothblum). All imaging experiments were performed at the Whitehead Institute Microscopy Facility. BK Kennedy is supported by a Leukemia Society of America Postdoctoral Fellowship. Dr Muramatsu of the Saitama Medical School of Japan, Department of Biochemistry graciously provided the original sample of PAF53 antisera used in these studies