Conditional defect in mRNA 3' end processing caused by a mutation in the gene for poly(A) polymerase.

Maturation of most eukaryotic mRNA 3' ends requires endonucleolytic cleavage and polyadenylation of precursor mRNAs. To further understand the mechanism and function of mRNA 3' end processing, we identified a temperature-sensitive mutant of Saccharomyces cerevisiae defective for polyadenylation. Genetic analysis showed that the polyadenylation defect and the temperature sensitivity for growth result from a single mutation. Biochemical analysis of extracts from this mutant shows that the polyadenylation defect occurs at a step following normal site-specific cleavage of a pre-mRNA at its polyadenylation site. Molecular cloning and characterization of the wild-type allele of the mutated gene revealed that it (PAP1) encodes a previously characterized poly(A) polymerase with unknown RNA substrate specificity. Analysis of mRNA levels and structure in vivo indicate that shift of growing, mutant cells to the nonpermissive temperature results in the production of poly(A)-deficient mRNAs which appear to end at their normal cleavage sites. Interestingly, measurement of the rate of protein synthesis after the temperature shift shows that translation continues long after the apparent loss of polyadenylated mRNA. Our characterization of the pap1-1 defect implicates this gene as essential for mRNA 3' end formation in S. cerevisiae.

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Functional characterization of human brown adipose tissue metabolism

Biochemical Journal ◽

10.1042/bcj20190464 ◽

2020 ◽

Vol 477 (7) ◽

pp. 1261-1286 ◽

Cited By ~ 3

Author(s):

Marie Anne Richard ◽

Hannah Pallubinsky ◽

Denis P. Blondin

Keyword(s):

Adipose Tissue ◽

Brown Adipose Tissue ◽

Functional Characterization ◽

Human Infants ◽

Positron Emission ◽

Brown Adipose ◽

Treatment Of Obesity ◽

And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

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DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

Biochemical Journal ◽

10.1042/bj20121085 ◽

2013 ◽

Vol 451 (3) ◽

pp. 453-461 ◽

Cited By ~ 26

Author(s):

Claudia C. S. Chini ◽

Carlos Escande ◽

Veronica Nin ◽

Eduardo N. Chini

Keyword(s):

Breast Cancer ◽

Nuclear Receptor ◽

Clock Genes ◽

Mrna Levels ◽

Protein Levels ◽

The Stability ◽

And Function ◽

Interfering Rna ◽

In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

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Characterization of a Temperature-Sensitive Vertebrate Clathrin Heavy Chain Mutant as a Tool to Study Clathrin-Dependent Events In Vivo

PLoS ONE ◽

10.1371/journal.pone.0012017 ◽

2010 ◽

Vol 5 (8) ◽

pp. e12017 ◽

Cited By ~ 3

Author(s):

Petra Neumann-Staubitz ◽

Stephanie L. Hall ◽

Joseph Kuo ◽

Antony P. Jackson

Keyword(s):

Heavy Chain ◽

Temperature Sensitive ◽

Clathrin Heavy Chain

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Isolation and characterization of a variant myoblast cell line that is temperature sensitive for differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2335-2341.1988 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2335-2341

Author(s):

R J Akhurst ◽

N B Flavin ◽

J Worden

Keyword(s):

Cell Line ◽

Progenitor Cell ◽

Temperature Shift ◽

Temperature Sensitive ◽

Isolation And Characterization ◽

New Variant ◽

Myoblast Cell Line ◽

Myoblast Cell ◽

Progenitor Cell Line

A new variant rat myogenic cell line, ts485, was isolated by subcloning the cell line ts3b2 (H. T. Nguyen, R. M. Medford, and B. Nadal-Ginard, Cell 34:281-293, 1983). Unlike the progenitor cell line, ts485 was thermosensitive for differentiation. Experiments with conditioned medium suggested that diffusible extracellular factors were not involved in dictating the differential phenotypes of ts485 cells cultured at the permissive and nonpermissive temperatures. Temperature shift experiments performed on cultures of ts485 cells indicated that the temperature-sensitive lesion was in a factor active during the growth phase and required to trigger a cascade of events leading to terminal differentiation.

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Chromatin structure and function: the heretical path to an RNA transcription factor

Biochemistry and Cell Biology ◽

10.1139/o96-067 ◽

1996 ◽

Vol 74 (5) ◽

pp. 623-632 ◽

Cited By ~ 5

Author(s):

Margarida O. Krause

Keyword(s):

Chromatin Structure ◽

Mammalian Cells ◽

T Antigen ◽

Temperature Sensitive ◽

Mobility Shift ◽

Cell Extracts ◽

Myc Gene ◽

And Function

This review represents a synthesis of the work of the author and her collaborators through 40 years of research aimed at an understanding of chromatin composition and functional arrangement. It describes the progressive experimental stages, starting with autoradiography and protein analysis and continuing on to a more functional approach testing the template properties of intact nuclei, as well as nuclei depleted of, or reconstituted with, defined fractions extracted from the chromatin of other cell lines or tissues. As new questions were raised at each phase of these studies, the investigation was shifted from chromosomal proteins to the role of a small RNA that coextracted with one protein fraction and whose properties suggested a transcription-activating function. The active RNA was identified as a class in RNA, designated as 7 SK. Its properties suggested a role in the activation of two oncogenes, the SV 40 T-antigen and the mammalian c-myc gene. A detailed analysis of the c-myc gene expression during transformation induction in temperature-sensitive mammalian cells finally culminated in in vivo evidence for a role of 7 SK in c-myc deregulation, using cells transfected with antisense oligonucleotides to block 7 SK activity. This was followed by an investigation of promoter targeting by 7 SK RNP using electrophoretic mobility shift assays with whole or 7 SK-depleted cell extracts. Taken together, these studies indicate that 7 SK RNP participates in transformation-dependent deregulation of the c-myc gene by activation of two c-myc minor promoters. The implications of these findings are discussed.Key words: chromatin structure, histones, nonhistones, 7 SK RNA, the c-myc gene, transcription regulation, SV 40, transformation.

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Isolation and characterization of a variant myoblast cell line that is temperature sensitive for differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2335 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2335-2341 ◽

Cited By ~ 4

Author(s):

R J Akhurst ◽

N B Flavin ◽

J Worden

Keyword(s):

Cell Line ◽

Progenitor Cell ◽

Temperature Shift ◽

Temperature Sensitive ◽

Isolation And Characterization ◽

New Variant ◽

Myoblast Cell Line ◽

Myoblast Cell ◽

Progenitor Cell Line

A new variant rat myogenic cell line, ts485, was isolated by subcloning the cell line ts3b2 (H. T. Nguyen, R. M. Medford, and B. Nadal-Ginard, Cell 34:281-293, 1983). Unlike the progenitor cell line, ts485 was thermosensitive for differentiation. Experiments with conditioned medium suggested that diffusible extracellular factors were not involved in dictating the differential phenotypes of ts485 cells cultured at the permissive and nonpermissive temperatures. Temperature shift experiments performed on cultures of ts485 cells indicated that the temperature-sensitive lesion was in a factor active during the growth phase and required to trigger a cascade of events leading to terminal differentiation.

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CHARACTERIZATION OF TEMPERATURE-SENSITIVE MUTANTS OF MEASLES VIRUS: TEMPERATURE-SHIFT EXPERIMENT

Japanese Journal of Medical Science and Biology ◽

10.7883/yoken1952.28.223 ◽

1975 ◽

Vol 28 (4) ◽

pp. 223-229 ◽

Cited By ~ 4

Author(s):

YUKIO YAMAZI ◽

FRANCIS L. BLACK ◽

HIROSHI HONDA ◽

YUKO TODOME ◽

MASARU SUGANUMA ◽

...

Keyword(s):

Measles Virus ◽

Temperature Shift ◽

Temperature Sensitive ◽

Temperature Sensitive Mutants ◽

Shift Experiment

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Nanomechanics and co-transcriptional folding of Spinach and Mango

Nature Communications ◽

10.1038/s41467-019-12299-y ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Jaba Mitra ◽

Taekjip Ha

Keyword(s):

Single Molecule ◽

Rna Aptamers ◽

Biophysical Characterization ◽

Mechanical Responses ◽

Discrete Step ◽

Force Relaxation ◽

G Quadruplex ◽

And Function

Abstract Recent advances in fluorogen-binding “light-up” RNA aptamers have enabled protein-free detection of RNA in cells. Detailed biophysical characterization of folding of G-Quadruplex (GQ)-based light-up aptamers such as Spinach, Mango and Corn is still lacking despite the potential implications on their folding and function. In this work we employ single-molecule fluorescence-force spectroscopy to examine mechanical responses of Spinach2, iMangoIII and MangoIV. Spinach2 unfolds in four discrete steps as force is increased to 7 pN and refolds in reciprocal steps upon force relaxation. In contrast, GQ-core unfolding in iMangoIII and MangoIV occurs in one discrete step at forces >10 pN and refolding occurred at lower forces showing hysteresis. Co-transcriptional folding using superhelicases shows reduced misfolding propensity and allowed a folding pathway different from refolding. Under physiologically relevant pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold. Understanding of the dynamics of RNA aptamers will aid engineering of improved fluorogenic modules for cellular applications.

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