Functional engagement of RNA polymerase II (Pol II) with eukaryotic chromosomes is a fundamental and highly regulated biological process. Here we present a high-resolution map of Pol II occupancy ...across the entire yeast genome. We compared a wild-type strain with a strain bearing a substitution in the Sen1 helicase, which is a Pol II termination factor for noncoding RNA genes. The wild-type pattern of Pol II distribution provides unexpected insights into the mechanisms by which genes are repressed or silenced. Remarkably, a single amino acid substitution that compromises Sen1 function causes profound changes in Pol II distribution over both noncoding and protein-coding genes, establishing an important function of Sen1 in the regulation of transcription. Given the strong similarity of the yeast and human Sen1 proteins, our results suggest that progressive neurological disorders caused by substitutions in the human Sen1 homolog Senataxin may be due to misregulation of transcription.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A eukaryotic chromosome contains many genes, each transcribed separately by RNA polymerase (pol) I, II or III. Transcription termination between genes prevents the formation of polycistronic RNAs and ...anti-sense RNAs, which are generally detrimental to the correct expression of genes. Terminating the transcription of protein-coding genes by pol II requires a group of proteins that also direct cleavage and polyadenylation of the messenger RNA in response to a specific sequence element, and are associated with the carboxyl-terminal domain of the largest subunit of pol II (refs 1, 2, 3, 4, 5, 6). By contrast, the cis-acting elements and trans-acting factors that direct termination of non-polyadenylated transcripts made by pol II, including small nucleolar and small nuclear RNAs, are not known. Here we show that read-through transcription from yeast small nucleolar RNA and small nuclear RNA genes into adjacent genes is prevented by a cis-acting element that is recognized, in part, by the essential RNA-binding protein Nrd1. The RNA-binding protein Nab3, the putative RNA helicase Sen1, and the intact C-terminal domain of pol II are also required for efficient response to the element. The same proteins are required for maintaining normal levels of Nrd1 mRNA, indicating that these proteins may control elongation of a subset of mRNA transcripts.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Forbs are a culturally and ecologically important yet poorly studied component of the Pacific Northwest Bunchgrass (PNB) ecosystem of western North America. Because many PNB forbs emerge early in the ...spring and senesce at the onset of summer, typical vegetation surveys, which occur mid‐summer, likely underrepresent forbs. We evaluated how the timing of vegetation sampling affected estimates of perennial forb abundance, richness, and floral density, with a focus on species that are culturally significant to Indigenous people of the region. We sampled 29 plots three times: mid‐summer (July), as well as 7 (May) and 12 (April) weeks prior. Timing had large effects on estimates, with significant declines in forb richness (42%), cover (80%), and floral density (95%) between April and July. The density of Camassia quamash and Lomatium cous, two culturally important species, declined 91% and 96%, respectively, from April to July. Nearly 65% of forbs had documented uses by Plateau Tribes. The consequences of biased estimates are significant and (1) limit our understanding of fundamental ecological processes and interactions; (2) hinder effective conservation and management because we lack information on forb abundance, status, and trends, and (3) result in disproportionately negative impacts to Indigenous communities as many forbs are important for subsistence and ceremonial purposes. Vegetation sampling during the spring months coincident with peak forb abundance, in addition to summer sampling would provide more appropriate data with which to evaluate and characterize PNB vegetation dynamics and improve our understanding, management, and conservation of native forbs.
We evaluated how the timing of vegetation sampling within the Pacific Northwest Bunchgrass ecosystem affected estimates of perennial forb abundance, richness, and floral density, with a focus on species of cultural significance to Indigenous people of the region. We found timing had a large effect on estimates, with significant declines in forb richness (42%), cover (80%), and floral density (95%) between April and July. The consequences of biased estimates: (1) limit our understanding of fundamental ecological processes and interactions; (2) hinder effective conservation and management because we lack information on forb abundance, status and trends, and (3) have a disproportionate negative impact on Indigenous communities as many forbs are important for subsistence and ceremonial purposes.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its ...structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276 nm, 377 nm, and 460 nm and a single emission peak at 530 nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We describe an architecture for organizing, integrating and sharing neurophysiology data within a single laboratory or across a group of collaborators. It comprises a database linking data files to ...metadata and electronic laboratory notes; a module collecting data from multiple laboratories into one location; a protocol for searching and sharing data and a module for automatic analyses that populates a website. These modules can be used together or individually, by single laboratories or worldwide collaborations.
Recent evidence suggests a role for the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) in pre-mRNA processing. The yeast NRD1 gene encodes an essential ...RNA-binding protein that shares homology with mammalian CTD-binding proteins and is thought to regulate mRNA abundance by binding to a specific cis-acting element. The present work demonstrates genetic and physical interactions among Nrd1p, the pol II CTD, Nab3p, and the CTD kinase CTDK-I. Previous studies have shown that Nrd1p associates with the CTD of pol II in yeast two-hybrid assays via its CTD-interaction domain (CID). We show that nrd1 temperature-sensitive alleles are synthetically lethal with truncation of the CTD to 9 or 10 repeats. Nab3p, a yeast hnRNP, is a high-copy suppressor of some nrd1 temperature-sensitive alleles, interacts with Nrd1p in a yeast two-hybrid assay, and coimmunoprecipitates with Nrd1p. Temperature-sensitive alleles of NAB3 are suppressed by deletion of CTK1, a kinase that has been shown to phosphorylate the CTD and increase elongation efficiency in vitro. This set of genetic and physical interactions suggests a role for yeast RNA-binding proteins in transcriptional regulation.
Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its ...amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3'-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3'-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
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