scholarly journals Long-read sequencing of nascent RNA reveals coupling among RNA processing events

2017 ◽  
Author(s):  
Lydia Herzel ◽  
Korinna Straube ◽  
Karla M. Neugebauer
Keyword(s):  
De Novo ◽  
Temperature Sensitive ◽  
Mrna Levels ◽  
Nascent Transcript ◽  
Long Read ◽  
Constitutive Splicing ◽  
Nuclear Exosome ◽  
Nascent Transcripts ◽  
Mrna Half Life ◽  
Regulatory Significance

AbstractPre-mRNA splicing is accomplished by the spliceosome, a megadalton complex that assembles de novo on each intron. Because spliceosome assembly and catalysis occur co-transcriptionally, we hypothesized that introns are removed in the order of their transcription in genomes dominated by constitutive splicing. Remarkably little is known about splicing order and the regulatory potential of nascent transcript remodeling by splicing, due to the limitations of existing methods that focus on analysis of mature splicing products (mRNAs) rather than substrates and intermediates. Here, we overcome this obstacle through long-read RNA sequencing of nascent, multi-intron transcripts in the fission yeast Schizosaccharomyces pombe. Most multi-intron transcripts were fully spliced, consistent with rapid co-transcriptional splicing. However, an unexpectedly high proportion of transcripts were either fully spliced or fully unspliced, suggesting that splicing of any given intron is dependent on the splicing status of other introns in the transcript. Supporting this, mild inhibition of splicing by a temperature-sensitive mutation in Prp2, the homolog of vertebrate U2AF65, increased the frequency of fully unspliced transcripts. Importantly, fully unspliced transcripts displayed transcriptional read-through at the polyA site and were degraded co-transcriptionally by the nuclear exosome. Finally, we show that cellular mRNA levels were reduced in genes with a high number of unspliced nascent transcripts during caffeine treatment, showing regulatory significance of co-transcriptional splicing. Therefore, overall splicing of individual nascent transcripts, 3’ end formation, and mRNA half-life depend on the splicing status of neighboring introns, suggesting crosstalk among spliceosomes and the polyA cleavage machinery during transcription elongation.

scholarly journals The Paf1 complex broadly impacts the transcriptome ofSaccharomyces cerevisiae

2019 ◽  
Author(s):  
Mitchell A. Ellison ◽  
Alex R. Lederer ◽  
Marcie H. Warner ◽  
Travis Mavrich ◽  
Elizabeth A. Raupach ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Differential Expression Analysis ◽  
Chromatin Modification ◽  
Antisense Transcription ◽  
Yeast Genome ◽  
Mrna Levels ◽  
Nascent Transcript ◽  
Protein Coding ◽  
Multiple Loci

ABSTRACTThe Polymerase Associated Factor 1 complex (Paf1C) is a multifunctional regulator of eukaryotic gene expression important for the coordination of transcription with chromatin modification and post-transcriptional processes. In this study, we investigated the extent to which the functions of Paf1C combine to regulate theSaccharomyces cerevisiaetranscriptome. While previous studies focused on the roles of Paf1C in controlling mRNA levels, here we took advantage of a genetic background that enriches for unstable transcripts and demonstrate that deletion ofPAF1affects all classes of Pol II transcripts including multiple classes of noncoding RNAs. By conducting ade novodifferential expression analysis independent of gene annotations, we found that Paf1 positively and negatively regulates antisense transcription at multiple loci. Comparisons with nascent transcript data revealed that many, but not all, changes in RNA levels detected by our analysis are due to changes in transcription instead of post-transcriptional events. To investigate the mechanisms by which Paf1 regulates protein-coding genes, we focused on genes involved in iron and phosphate homeostasis, which were differentially affected byPAF1deletion. Our results indicate that Paf1 stimulates phosphate gene expression through a mechanism that is independent of any individual Paf1C-dependent histone modification. In contrast, the inhibition of iron gene expression by Paf1 correlates with a defect in H3 K36 tri-methylation. Finally, we showed that one iron regulon gene,FET4, is coordinately controlled by Paf1 and transcription of upstream noncoding DNA. Together these data identify roles for Paf1C in controlling both coding and noncoding regions of the yeast genome.

scholarly journals The Paf1 Complex Broadly Impacts the Transcriptome of Saccharomyces cerevisiae

Genetics ◽  
2019 ◽  
Vol 212 (3) ◽  
pp. 711-728 ◽  
Author(s):  
Mitchell A. Ellison ◽  
Alex R. Lederer ◽  
Marcie H. Warner ◽  
Travis N. Mavrich ◽  
Elizabeth A. Raupach ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Differential Expression Analysis ◽  
Chromatin Modification ◽  
Antisense Transcription ◽  
Yeast Genome ◽  
Mrna Levels ◽  
Nascent Transcript ◽  
Link Type

The Polymerase Associated Factor 1 complex (Paf1C) is a multifunctional regulator of eukaryotic gene expression important for the coordination of transcription with chromatin modification and post-transcriptional processes. In this study, we investigated the extent to which the functions of Paf1C combine to regulate the Saccharomyces cerevisiae transcriptome. While previous studies focused on the roles of Paf1C in controlling mRNA levels, here, we took advantage of a genetic background that enriches for unstable transcripts, and demonstrate that deletion of PAF1 affects all classes of Pol II transcripts including multiple classes of noncoding RNAs (ncRNAs). By conducting a de novo differential expression analysis independent of gene annotations, we found that Paf1 positively and negatively regulates antisense transcription at multiple loci. Comparisons with nascent transcript data revealed that many, but not all, changes in RNA levels detected by our analysis are due to changes in transcription instead of post-transcriptional events. To investigate the mechanisms by which Paf1 regulates protein-coding genes, we focused on genes involved in iron and phosphate homeostasis, which were differentially affected by PAF1 deletion. Our results indicate that Paf1 stimulates phosphate gene expression through a mechanism that is independent of any individual Paf1C-dependent histone modification. In contrast, the inhibition of iron gene expression by Paf1 correlates with a defect in H3 K36 trimethylation. Finally, we showed that one iron regulon gene, FET4, is coordinately controlled by Paf1 and transcription of upstream noncoding DNA. Together, these data identify roles for Paf1C in controlling both coding and noncoding regions of the yeast genome.

scholarly journals Direct Interactions with Nascent Transcripts Is Potentially a Common Targeting Mechanism of Long Non-Coding RNAs

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1483
Author(s):  
Ivan Antonov ◽  
Yulia Medvedeva
Keyword(s):  
Target Genes ◽  
Regulation Of Gene Expression ◽  
Pilot Project ◽  
Base Pairing ◽  
Nascent Transcript ◽  
Active Transcription ◽  
Wet Lab ◽  
In Trans ◽  
Non Coding Rnas ◽  
Nascent Transcripts

Although thousands of mammalian long non-coding RNAs (lncRNAs) have been reported in the last decade, their functional annotation remains limited. A wet-lab approach to detect functions of a novel lncRNA usually includes its knockdown followed by RNA sequencing and identification of the deferentially expressed genes. However, identification of the molecular mechanism(s) used by the lncRNA to regulate its targets frequently becomes a challenge. Previously, we developed the ASSA algorithm that detects statistically significant inter-molecular RNA-RNA interactions. Here we designed a workflow that uses ASSA predictions to estimate the ability of an lncRNA to function via direct base pairing with the target transcripts (co- or post-transcriptionally). The workflow was applied to 300+ lncRNA knockdown experiments from the FANTOM6 pilot project producing statistically significant predictions for 71 unique lncRNAs (104 knockdowns). Surprisingly, the majority of these lncRNAs were likely to function co-transcriptionally, i.e., hybridize with the nascent transcripts of the target genes. Moreover, a number of the obtained predictions were supported by independent iMARGI experimental data on co-localization of lncRNA and chromatin. We detected an evolutionarily conserved lncRNA CHASERR (AC013394.2 or LINC01578) that could regulate target genes co-transcriptionally via interaction with a nascent transcript by directing CHD2 helicase. The obtained results suggested that this nuclear lncRNA may be able to activate expression of the target genes in trans by base-pairing with the nascent transcripts and directing the CHD2 helicase to the regulated promoters leading to open the chromatin and active transcription. Our study highlights the possible importance of base-pairing between nuclear lncRNAs and nascent transcripts for the regulation of gene expression.

Abstract 112: Myeloid Mineralocorticoid Receptor Controls Inflammatory and Fibrotic Responses After Renal Ischemic Injury via Macrophage Interleukin-4 Receptor

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Jonatan Barrera-Chimal ◽  
Sebastian M Lechner ◽  
Soumaya E Moghrabi ◽  
Peter Kolkhof ◽  
Frédéric Jaisser
Keyword(s):  
Mineralocorticoid Receptor ◽  
De Novo ◽  
Interstitial Fibrosis ◽  
Macrophage Polarization ◽  
Peritoneal Macrophages ◽  
Renal Ischemia ◽  
Receptor Expression ◽  
Interleukin 4 ◽  
Mrna Levels ◽  
Kidney Fibrosis

Introduction: Patients who survive an episode of acute kidney injury (AKI) are at high risk of de novo chronic kidney disease (CKD) development. Pharmacological mineralocorticoid receptor (MR) antagonism is useful to prevent CKD after a single episode of ischemic AKI in the rat. Objective: Test the involvement of myeloid MR in the development of kidney fibrosis after an ischemic AKI episode. Methods: We included 18 male C57/B6 mice that were divided in: sham, renal ischemia for 22.5 min and IR plus treatment with the non-steroidal MR antagonist finerenone (10 mg/kg) at -48, -24 and -1 h before IR. MR inactivation in myeloid cells (MR MyKO ) was achieved by crossing mice with the MR alleles flanked by loxP sites (MR f/f ) with mice expressing the Cre recombinase under the LysM promoter activity. In MR f/f and MR MyKO mice we induced renal IR of 22.5 min or sham surgery. The mice were followed-up during 4 weeks to test for AKI to CKD transition. In another set of mice, the macrophages were sorted from kidneys after 24 h of reperfusion and flow cytometry characterization or mRNA extraction was performed. Thyoglycolate elicited peritoneal macrophages were used for in vitro studies. Results: The progression of AKI to CKD after 4 weeks of renal ischemia in the untreated C57/B6 and MR f/f mice was characterized by a 50% increase in plasma creatinine, a 2-fold increase in the mRNA levels of TGF-β and fibronectin as well as by severe tubule-interstitial fibrosis. The mice that received finerenone or MR MyKO mice were protected against these alterations. Increased expression of M2-anti-inflamatory markers in kidney-isolated macrophages from finerenone-treated or MR MyKO mice was observed. The inflammatory population of Ly6C high macrophages was reduced by 50%. In peritoneal macrophages in culture, MR inhibition promoted increased IL-4 receptor expression and activation, facilitating macrophage polarization to an M2 phenotype. Conclusion: MR antagonism or myeloid MR deficiency facilitates macrophage polarization to a M2, anti-inflammatory phenotype after kidney IR, preventing maladaptive repair and chronic kidney fibrosis and dysfunction. MR inhibition acts through the modulation of IL-4 receptor signaling to facilitate macrophage phenotype switching.

Growth hormone decreases muscle glutamine production and stimulates protein synthesis in hypercatabolic patients

2000 ◽  
Vol 279 (2) ◽  
pp. E323-E332 ◽  
Author(s):  
Gianni Biolo ◽  
Fulvio Iscra ◽  
Alessandra Bosutti ◽  
Gabriele Toigo ◽  
Beniamino Ciocchi ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Protein Synthesis ◽  
Amino Acid ◽  
De Novo ◽  
Muscle Protein ◽  
Recombinant Human Growth Hormone ◽  
Glutamine Metabolism ◽  
Trauma Patients ◽  
Mrna Levels ◽  
Glutamine Production

We determined the effects of 24-h recombinant human growth hormone (rhGH) infusion into a femoral artery on leg muscle protein kinetics, amino acid transport, and glutamine metabolism in eight adult hypercatabolic trauma patients. Metabolic pathways were assessed by leg arteriovenous catheterization and muscle biopsies with the use of stable amino acid isotopes. Muscle mRNA levels of selected enzymes were determined by competitive PCR. rhGH infusion significantly accelerated the inward transport rates of phenylalanine and leucine and protein synthesis, whereas the muscle protein degradation rate and cathepsin B and UbB polyubiquitin mRNA levels were not significantly modified by rhGH. rhGH infusion decreased the rate of glutamine de novo synthesis and glutamine precursor availability, total branched-chain amino acid catabolism, and nonprotein glutamate utilization. Thus net glutamine release from muscle into circulation significantly decreased after rhGH administration (∼50%), whereas glutamine synthetase mRNA levels increased after rhGH infusion, possibly to compensate for reduced glutamine precursor availability. We conclude that, after trauma, the anticatabolic action of rhGH is associated with a potentially harmful decrease in muscle glutamine production.

Long‐read sequencing and de novo assembly of the Luffa cylindrica (L.) Roem. genome

2020 ◽  
Vol 20 (2) ◽  
pp. 511-519 ◽  
Author(s):  
Tao Zhang ◽  
Xuyan Ren ◽  
Zhao Zhang ◽  
Yao Ming ◽  
Zhe Yang ◽  
...  
Keyword(s):  
De Novo Assembly ◽  
De Novo ◽  
Luffa Cylindrica ◽  
Long Read

scholarly journals Metabolic Effects of Oral Phenelzine Treatment on High-Sucrose-Drinking Mice

2018 ◽  
Vol 19 (10) ◽  
pp. 2904 ◽  
Author(s):  
Christian Carpéné ◽  
Saioa Gómez-Zorita ◽  
Alice Chaplin ◽  
Josep Mercader
Keyword(s):  
Adipose Tissue ◽  
Phosphoenolpyruvate Carboxykinase ◽  
De Novo ◽  
Uncoupling Protein ◽  
De Novo Lipogenesis ◽  
Metabolic Effects ◽  
Mrna Levels ◽  
Brown Adipose ◽  
High Sucrose ◽  
Sucrose Drinking

Phenelzine has been suggested to have an antiobesity effect by inhibiting de novo lipogenesis, which led us to investigate the metabolic effects of oral chronic phenelzine treatment in high-sucrose-drinking mice. Sucrose-drinking mice presented higher body weight gain and adiposity versus controls. Phenelzine addition did not decrease such parameters, even though fat pad lipid content and weights were not different from controls. In visceral adipocytes, phenelzine did not impair insulin-stimulated de novo lipogenesis and had no effect on lipolysis. However, phenelzine reduced the mRNA levels of glucose transporters 1 and 4 and phosphoenolpyruvate carboxykinase in inguinal white adipose tissue (iWAT), and altered circulating levels of free fatty acids (FFA) and glycerol. Interestingly, glycemia was restored in phenelzine-treated mice, which also had higher insulinaemia. Phenelzine-treated mice presented higher rectal temperature, which was associated to reduced mRNA levels of uncoupling protein 1 in brown adipose tissue. Furthermore, unlike sucrose-drinking mice, hepatic malondialdehyde levels were not altered. In conclusion, although de novo lipogenesis was not inhibited by phenelzine, the data suggest that the ability to re-esterify FFA is impaired in iWAT. Moreover, the effects on glucose homeostasis and oxidative stress suggest that phenelzine could alleviate obesity-related alterations and deserves further investigation in obesity models.

Growth-dependent regulation of system A in SV40-transformed fetal rat hepatocytes

1988 ◽  
Vol 255 (3) ◽  
pp. C261-C270 ◽  
Author(s):  
M. E. Handlogten ◽  
M. S. Kilberg
Keyword(s):  
Cell Density ◽  
De Novo ◽  
Membrane Vesicles ◽  
Rat Hepatocytes ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
System A

Fetal RLA209-15 hepatocytes, transformed with a temperature-sensitive SV40 mutant, behave like fully differentiated cells at the growth-restrictive temperature of 40 degrees C. Conversely, incubation at the growth-permissive temperature of 33 degrees C results in a transformed phenotype characterized by rapid cell division and decreased production of liver-specific proteins. The results presented here demonstrate that the cells at 33 degrees C exhibited high rates of system A transport, but transfer to 40 degrees C reduced the activity greater than 50% within 24 h. This decline in transport was independent of cell density, although the basal rate of uptake was inversely proportional to cell density in rapidly dividing cells. Transfer of cells from 40 to 33 degrees C resulted in an enhancement of system A activity that was blocked by tunicamycin. Plasma membrane vesicles from cells maintained at either 33 or 40 degrees C retained uptake rates proportional to those in the intact cells; this difference in transport activity could also be demonstrated after detergent solubilization and reconstitution. Collectively, these data indicate that de novo synthesis of the system A carrier is regulated in conjunction with temperature-dependent cell growth in RLA209-15 hepatocytes.

scholarly journals Genotyping structural variants in pangenome graphs using the vg toolkit

2019 ◽  
Author(s):  
Glenn Hickey ◽  
David Heller ◽  
Jean Monlong ◽  
Jonas A. Sibbesen ◽  
Jouni Sirén ◽  
...  
Keyword(s):  
De Novo ◽  
State Of The Art ◽  
Effective Means ◽  
Point Mutations ◽  
Structural Variants ◽  
Short Read ◽  
Yeast Strains ◽  
Sequencing Studies ◽  
Long Read

AbstractStructural variants (SVs) remain challenging to represent and study relative to point mutations despite their demonstrated importance. We show that variation graphs, as implemented in the vg toolkit, provide an effective means for leveraging SV catalogs for short-read SV genotyping experiments. We benchmarked vg against state-of-the-art SV genotypers using three sequence-resolved SV catalogs generated by recent long-read sequencing studies. In addition, we use assemblies from 12 yeast strains to show that graphs constructed directly from aligned de novo assemblies improve genotyping compared to graphs built from intermediate SV catalogs in the VCF format.

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