scholarly journals An ortholog of the Vasa intronic gene is required for small RNA-mediated translation repression inChlamydomonas reinhardtii

2019 ◽  
Vol 117 (1) ◽  
pp. 761-770 ◽  
Author(s):  
Xinrong Ma ◽  
Fadia Ibrahim ◽  
Eun-Jeong Kim ◽  
Scott Shaver ◽  
James Becker ◽  
...  
Keyword(s):  
Small Rnas ◽  
Messenger Rna ◽  
Sucrose Density Gradient ◽  
Rna Degradation ◽  
Translation Elongation ◽  
Translation Inhibition ◽  
Translation Repression ◽  
Mrna Destabilization ◽  
Sucrose Density Gradient Sedimentation ◽  
Density Gradient Sedimentation

Small RNAs (sRNAs) associate with Argonaute (AGO) proteins in effector complexes, termed RNA-induced silencing complexes (RISCs), which regulate complementary transcripts by translation inhibition and/or RNA degradation. In the unicellular algaChlamydomonas, several metazoans, and land plants, emerging evidence indicates that polyribosome-associated transcripts can be translationally repressed by RISCs without substantial messenger RNA (mRNA) destabilization. However, the mechanism of translation inhibition in a polyribosomal context is not understood. Here we show thatChlamydomonasVIG1, an ortholog of theDrosophila melanogasterVasa intronic gene (VIG), is required for this process. VIG1 localizes predominantly in the cytosol and comigrates with monoribosomes and polyribosomes by sucrose density gradient sedimentation. AVIG1-deleted mutant shows hypersensitivity to the translation elongation inhibitor cycloheximide, suggesting that VIG1 may have a nonessential role in ribosome function/structure. Additionally, FLAG-tagged VIG1 copurifies with AGO3 and Dicer-like 3 (DCL3), consistent with it also being a component of the RISC. Indeed, VIG1 is necessary for the repression of sRNA-targeted transcripts at the translational level but is dispensable for cleavage-mediated RNA interference and for the association of the AGO3 effector with polyribosomes or target transcripts. Our results suggest that VIG1 is an ancillary ribosomal component and plays a role in sRNA-mediated translation repression of polyribosomal transcripts.

scholarly journals Evidence for 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats

1982 ◽  
Vol 204 (1) ◽  
pp. 31-36 ◽  
Author(s):  
D Sömjen ◽  
G J Sömjen ◽  
Y Weisman ◽  
I Binderman
Keyword(s):  
Density Gradient ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Macromolecular Complex ◽  
Long Bones ◽  
Newborn Rats ◽  
Sucrose Density Gradient Sedimentation ◽  
Density Gradient Sedimentation ◽  
Binding Component

Several reports have appeared that suggest that 24,25-dihydroxycholecalciferol has a possible biological role in bone formation. We have utilized competition studies, saturation analysis, sucrose-density-gradient sedimentation and DEAE-cellulose chromatography to demonstrate that long bones of vitamin D-depleted newborn rats contain cytoplasmic and possibly nuclear receptors that bind 24,25-dihydroxycholecalciferol with specificity and high affinity (Kd = 1.79 nM). Sucrose-density-gradient analysis of the cytoplasmic 24,25-dihydroxycholecalciferol-binding component showed a single binding macromolecule for 24,25-dihydroxycholecalciferol with a sedimentation coefficient of 3.1 S. DEAE-cellulose chromatography showed a [3H]24,25, dihydroxycholecalciferol-macromolecular complex that binds to DEAE-cellulose and elutes between 0.15 and 0.21 M-KCl. The finding of 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats suggests a possible involvement of 24,25-dihydroxycholecalciferol in the metabolism of developing skeletal tissues.

scholarly journals Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light-Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Jun Takezawa ◽  
Yukio Ishimi ◽  
Naomi Aiba ◽  
Kouichi Yamada
Keyword(s):  
Density Gradient ◽  
Hela Cells ◽  
Uv Light ◽  
Sucrose Density Gradient ◽  
Detectable Effect ◽  
Main Role ◽  
Template Strand ◽  
Apparent Evidence ◽  
Sucrose Density Gradient Sedimentation ◽  
Density Gradient Sedimentation

When a replicative DNA polymerase stalls upon encountering a lesion on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion, extend by a few nucleotides, and dissociate from the 3′-OH. The replicative polymerase then resumes DNA synthesis. This process, termed translesion replication (TLS) or replicative bypass, may involve at least five different polymerases in mammals, although the participating polymerases and their roles have not been entirely characterized. Using siRNAs originally designed and an alkaline sucrose density gradient sedimentation technique, we verified the involvement of several polymerases in ultraviolet (UV) light-induced TLS in HeLa cells. First, siRNAs to Rev3 or Rev7 largely abolished UV-TLS, suggesting that these 2 gene products, which comprise Polζ, play a main role in mutagenic TLS. Second, Rev1-targeted siRNA also abrogated UV-TLS, indicating that Rev1 is also indispensable to mutagenic TLS. Third, Polη-targeted siRNA also prevented TLS to a greater extent than our expectations. Forth, although siRNA to Polι had no detectable effect, that to Polκ delayed UV-TLS. To our knowledge, this is the first study reporting apparent evidence for the participation of Polκ in UV-TLS.

Separation of paraoxon and mipafox sensitive esterases by sucrose density gradient sedimentation

1983 ◽  
Vol 17 (3-4) ◽  
pp. 315-320 ◽  
Author(s):  
Yuji Ishikawa ◽  
Edward Chow ◽  
Mark G. McNamee ◽  
Michael McChesney ◽  
Barry W. Wilson
Keyword(s):  
Density Gradient ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Sedimentation ◽  
Density Gradient Sedimentation

scholarly journals The radish Ogura fertility restorer impedes translation elongation along its cognate CMS-causing mRNA

2021 ◽  
Vol 118 (35) ◽  
pp. e2105274118
Author(s):  
Chuande Wang ◽  
Lina Lezhneva ◽  
Nadège Arnal ◽  
Martine Quadrado ◽  
Hakim Mireau
Keyword(s):  
Messenger Rna ◽  
Mrna Translation ◽  
Pollen Sterility ◽  
Genetic Interactions ◽  
Gene Control ◽  
Fertility Restorer ◽  
Translation Elongation ◽  
Translation Inhibition ◽  
Restorer Of Fertility ◽  
Fertility Restorers

The control of messenger RNA (mRNA) translation has been increasingly recognized as a key regulatory step for gene control, but clear examples in eukaryotes are still scarce. Nucleo-cytoplasmic male sterilities (CMS) represent ideal genetic models to dissect genetic interactions between the mitochondria and the nucleus in plants. This trait is determined by specific mitochondrial genes and is associated with a pollen sterility phenotype that can be suppressed by nuclear genes known as restorer-of-fertility (Rf). In this study, we focused on the Ogura CMS system in rapeseed and showed that reversion to male sterility by the PPR-B fertility restorer (also called Rfo) occurs through a specific translation inhibition of the mitochondria-encoded CMS-causing mRNA orf138. We also demonstrate that PPR-B binds within the coding sequence of orf138 and acts as a ribosome blocker to specifically impede translation elongation along the orf138 mRNA. Rfo is the first recognized fertility restorer shown to act this way. These observations will certainly facilitate the development of synthetic fertility restorers for CMS systems in which efficient natural Rfs are lacking.

scholarly journals Control of sarcoplasmic/endoplasmic-reticulum Ca2+ pump expression in cardiac and smooth muscle

1999 ◽  
Vol 338 (1) ◽  
pp. 167-173 ◽  
Author(s):  
Christine M. MISQUITTA ◽  
Angela SING ◽  
Ashok K. GROVER
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Cardiac Muscle ◽  
Sucrose Density Gradient ◽  
Sedimentation Velocity ◽  
Translation Efficiency ◽  
Mrna Levels ◽  
Sedimentation Pattern ◽  
Sucrose Density Gradient Sedimentation ◽  
Density Gradient Sedimentation

Cardiac muscle expresses sarcoplasmic/endoplasmic-reticulum Ca2+ pump isoform SERCA2a; stomach smooth muscle expresses SERCA2b. In 2-day-old rabbits, cardiac muscle contained levels of SERCA2 protein that were 100–200-fold those in the stomach smooth muscle. In nuclear run-on assays, the rate of SERCA2 gene transcription in heart nuclei was not significantly higher than in the stomach smooth-muscle nuclei. However, the SERCA2 mRNA levels (mean±S.E.M.) were (29±4)-fold higher in the heart. In both tissues the SERCA2 mRNA was associated with polyribosomes. In a sucrose-density-gradient sedimentation velocity experiment on polyribosomes, there was no difference in the sedimentation pattern of SERCA2 mRNA between the two tissues, suggesting that the translation efficiency of SERCA2 RNA in the two tissues is quite similar. Thus the main difference in the control of SERCA2 expression in the two tissues is post-transcriptional and pretranslational.

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