scholarly journals Polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression in mycobacteria

2019 ◽  
Author(s):  
Jill G. Canestrari ◽  
Erica Lasek-Nesselquist ◽  
Ashutosh Upadhyay ◽  
Martina Rofaeil ◽  
Matthew M. Champion ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mutational Analysis ◽  
Open Reading Frames ◽  
Level Control ◽  
Small Proteins ◽  
Short Orfs ◽  
The Many ◽  
Ribosome Pausing ◽  
Proteomic Responses ◽  
Open Question

ABSTRACTGenome-wide transcriptomic analyses have revealed abundant expressed short open reading frames (ORFs) in bacteria. Whether these short ORFs, or the small proteins they encode, are functional remains an open question. One quarter of mycobacterial mRNAs are leaderless, beginning with a 5’-AUG or GUG initiation codon. Leaderless mRNAs often encode unannotated short ORFs as the first gene of a polycistronic transcript. Here we show that polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression. Detailed mutational analysis shows that one polycysteine short ORF controls expression of the downstream genes. Our data indicate that ribosomes stalled in the polycysteine tract block mRNA structures that otherwise sequester the ribosome-binding site of the 3’gene. We assessed endogenous proteomic responses to cysteine limitation in Mycobacterium smegmatis using mass spectrometry. Six cysteine metabolic loci having unannotated polycysteine-encoding leaderless short ORF architectures responded to cysteine limitation, revealing widespread cysteine-responsive attenuation in mycobacteria. Individual leaderless short ORFs confer independent operon-level control, while their shared dependence on cysteine ensures a collective response mediated by ribosome pausing. We propose the term ribulon to classify ribosome-directed regulons. Regulon-level coordination by ribosomes on sensory short ORFs illustrates one utility of the many unannotated short ORFs expressed in bacterial genomes.

scholarly journals Identification of novel translated small ORFs in Escherichia coli using complementary ribosome profiling approaches

2021 ◽  
Author(s):  
Anne M Stringer ◽  
Carol Smith ◽  
Kyle Mangano ◽  
Joseph Thomas Wade
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
High Sensitivity ◽  
Purifying Selection ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Small Subset ◽  
Stop Codons ◽  
Small Proteins ◽  
Short Orfs

Small proteins of <51 amino acids are abundant across all domains of life but are often overlooked because their small size makes them difficult to predict computationally, and they are refractory to standard proteomic approaches. Ribosome profiling has been used to infer the existence of small proteins by detecting the translation of the corresponding open reading frames (ORFs). Detection of translated short ORFs by ribosome profiling can be improved by treating cells with drugs that stall ribosomes at specific codons. Here, we combine the analysis of ribosome profiling data for Escherichia coli cells treated with antibiotics that stall ribosomes at either start or stop codons. Thus, we identify ribosome-occupied start and stop codons for ~400 novel putative ORFs with high sensitivity. The newly discovered ORFs are mostly short, with 365 encoding proteins of <51 amino acids. We validate translation of several selected short ORFs, and show that many likely encode unstable proteins. Moreover, we present evidence that most of the newly identified short ORFs are not under purifying selection, suggesting they do not impact cell fitness, although a small subset have the hallmarks of functional ORFs.

scholarly journals Sight of parasitoid wasps accelerates sexual behavior and upregulates a micropeptide gene in Drosophila

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shimaa A. M. Ebrahim ◽  
Gaëlle J. S. Talross ◽  
John R. Carlson
Keyword(s):  
Gene Expression ◽  
Nervous System ◽  
Sexual Behavior ◽  
Behavioral Response ◽  
Mutational Analysis ◽  
Parasitoid Wasp ◽  
Parasitoid Wasps ◽  
Wasp Species ◽  
Defensive Responses ◽  
Vast Range

AbstractParasitoid wasps inflict widespread death upon the insect world. Hundreds of thousands of parasitoid wasp species kill a vast range of insect species. Insects have evolved defensive responses to the threat of wasps, some cellular and some behavioral. Here we find an unexpected response of adult Drosophila to the presence of certain parasitoid wasps: accelerated mating behavior. Flies exposed to certain wasp species begin mating more quickly. The effect is mediated via changes in the behavior of the female fly and depends on visual perception. The sight of wasps induces the dramatic upregulation in the fly nervous system of a gene that encodes a 41-amino acid micropeptide. Mutational analysis reveals that the gene is essential to the behavioral response of the fly. Our work provides a foundation for further exploration of how the activation of visual circuits by the sight of a wasp alters both sexual behavior and gene expression.

scholarly journals A First Step towards Learning which uORFs Regulate Gene Expression

2006 ◽  
Vol 3 (2) ◽  
pp. 109-122 ◽  
Author(s):  
◽  
Christopher H. Bryant ◽  
Graham J.L. Kemp ◽  
Marija Cvijovic
Keyword(s):  
Gene Expression ◽  
Inductive Logic ◽  
Preliminary Evidence ◽  
Open Reading Frames ◽  
Yeast Saccharomyces Cerevisiae ◽  
Regulate Gene Expression ◽  
Integrative System ◽  
Upstream Open Reading Frames ◽  
Reading Frames ◽  
Regulate Gene

Summary We have taken a first step towards learning which upstream Open Reading Frames (uORFs) regulate gene expression (i.e., which uORFs are functional) in the yeast Saccharomyces cerevisiae. We do this by integrating data from several resources and combining a bioinformatics tool, ORF Finder, with a machine learning technique, inductive logic programming (ILP). Here, we report the challenge of using ILP as part of this integrative system, in order to automatically generate a model that identifies functional uORFs. Our method makes searching for novel functional uORFs more efficient than random sampling. An attempt has been made to predict novel functional uORFs using our method. Some preliminary evidence that our model may be biologically meaningful is presented.

Analysis of a tissue-specific enhancer: ARF6 regulates adipogenic gene expression

1992 ◽  
Vol 12 (3) ◽  
pp. 1202-1208
Author(s):  
R A Graves ◽  
P Tontonoz ◽  
B M Spiegelman
Keyword(s):  
Gene Expression ◽  
Cultured Cells ◽  
Mutational Analysis ◽  
Cell Types ◽  
Specific Gene ◽  
Enhancer Activity ◽  
Cis Acting ◽  
Specific Gene Expression ◽  
Nuclear Extracts ◽  
Adipogenic Gene

The molecular basis of adipocyte-specific gene expression is not well understood. We have previously identified a 518-bp enhancer from the adipocyte P2 gene that stimulates adipose-specific gene expression in both cultured cells and transgenic mice. In this analysis of the enhancer, we have defined and characterized a 122-bp DNA fragment that directs differentiation-dependent gene expression in cultured preadipocytes and adipocytes. Several cis-acting elements have been identified and shown by mutational analysis to be important for full enhancer activity. One pair of sequences, ARE2 and ARE4, binds a nuclear factor (ARF2) present in extracts derived from many cell types. Multiple copies of these elements stimulate gene expression from a minimal promoter in preadipocytes, adipocytes, and several other cultured cell lines. A second pair of elements, ARE6 and ARE7, binds a separate factor (ARF6) that is detected only in nuclear extracts derived from adipocytes. The ability of multimers of ARE6 or ARE7 to stimulate promoter activity is strictly adipocyte specific. Mutations in the ARE6 sequence greatly reduce the activity of the 518-bp enhancer. These data demonstrate that several cis- and trans-acting components contribute to the activity of the adipocyte P2 enhancer and suggest that ARF6, a novel differentiation-dependent factor, may be a key regulator of adipogenic gene expression.

Alternative translation and alternative RNA processing mechanism in parvovirus RNA processing

2014 ◽  
Author(s):  
◽  
Olufemi Fasina
Keyword(s):  
Gene Expression ◽  
Rna Processing ◽  
Viral Genome ◽  
Transcription Initiation ◽  
Alternative Polyadenylation ◽  
Open Reading Frames ◽  
Genome Replication ◽  
University Of Missouri ◽  
Diverse Range ◽  
Viral Genome Replication

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Viruses as obligate intracellular metabolic parasite require the capacity to orchestrate and modulate the host environment either in the nucleus or cytoplasm for their efficient reproductive life cycle. This warrants the use of diverse range of proteins expressed from the viral genome with the ability of regulating viral genome replication, transcription and translation, in addition antagonizing host factors inhibitory to the virus. Therefore, in order to achieve these goals, viruses utilizes gene expression strategies to expand their coding capacity. Gene expression mechanism such as transcription initiation, capping, splicing and 3�-end processing afford viruses the opportunities to utilize the eukaryotic metabolic machineries for generating proteome diversity. Parvoviruses and other DNA viruses effectively capitalize on their use of nuclear eukaryotic metabolic machineries to co-opt host cell factors for optimal replication and gene expression. Parvoviruses with small genome size and overlapping open reading frames utilize alternative transcription initiation, alternative splicing and alternative polyadenylation to co-ordinate the expression of its non-structural and structural proteins. In this work, we have characterized how two parvoviruses; Dependovirus AAV5 and Bocavirus Minute virus of canine (MVC) utilize alternative gene expression mechanisms and strategies to optimize expression of viral proteins from their genome.

Mechanism of RSV-induced IL-8 gene expression in A549 cells before viral replication

1996 ◽  
Vol 271 (6) ◽  
pp. L963-L971 ◽  
Author(s):  
M. A. Fiedler ◽  
K. Wernke-Dollries ◽  
J. M. Stark
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Viral Replication ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
A549 Cells ◽  
Nf Kappa B ◽  
Mobility Shift ◽  
Syncytial Virus ◽  
Time Point

Previous studies demonstrated that respiratory syncytial virus (RSV) infection of A549 cells induced interleukin (IL)-8 gene expression and protein release from the cells as early as 2 h after treatment [M. A. Fiedler, K. Wernke-Dollries, and J. M. Stark. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L865-L872, 1995; J. G. Mastronarde, M. M. Monick, and G. W. Hunninghake. Am. J. Respir. Cell Mol. Biol. 13: 237-244, 1995]. Furthermore, the effects of RSV at the 2-h time point were not dependent on viral replication. The studies reported here were designed to test the hypothesis that active and inactive RSV induce IL-8 gene expression in A549 cells at the 2-h time point by a mechanism dependent on the activation of the nuclear transcription factor NF-kappa B Northern blot analysis indicated that IL-8 gene expression occurred independent of protein synthesis 2 h after A549 cells were treated with RSV. Analysis of nuclear extracts from RSV-treated A549 cells by electrophoretic mobility shift assays demonstrated that NF-kappa B was activated as early as 15 min after RSV was added to the cells and remained activated for at least 90 min. In contrast, baseline levels of NF-IL-6 and activator protein-1 (AP-1) did not change over this period of time. Deoxyribonuclease footprint analysis of a portion of the 5'-flanking region of the IL-8 gene demonstrated two potential regions for transcription factor binding, which corresponded to the potential AP-1 binding site, and potential NF-IL-6 and NF-kappa B binding sites. Mutational analysis of the 200-bp 5'-untranslated region of the IL-8 gene demonstrated that activation of NF-kappa B and NF-IL-6 were required for RSV-induced transcriptional activation of the IL-8 gene.

scholarly journals A scalable platform for the development of cell-type-specific viral drivers

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Sinisa Hrvatin ◽  
Christopher P Tzeng ◽  
M Aurel Nagy ◽  
Hume Stroud ◽  
Charalampia Koutsioumpa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
High Specificity ◽  
Cell Types ◽  
Regulatory Elements ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Cell Type Specific ◽  
The Many ◽  
Dna Regulatory Elements

Enhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression. Recent studies characterizing individual enhancers have revealed their potential to direct heterologous gene expression in a highly cell-type-specific manner. However, it has not yet been possible to systematically identify and test the function of enhancers for each of the many cell types in an organism. We have developed PESCA, a scalable and generalizable method that leverages ATAC- and single-cell RNA-sequencing protocols, to characterize cell-type-specific enhancers that should enable genetic access and perturbation of gene function across mammalian cell types. Focusing on the highly heterogeneous mammalian cerebral cortex, we apply PESCA to find enhancers and generate viral reagents capable of accessing and manipulating a subset of somatostatin-expressing cortical interneurons with high specificity. This study demonstrates the utility of this platform for developing new cell-type-specific viral reagents, with significant implications for both basic and translational research.

scholarly journals Identification of novel translated small ORFs in Escherichia coli using complementary ribosome profiling approaches

2021 ◽  
Author(s):  
Anne Stringer ◽  
Carol Smith ◽  
Kyle Mangano ◽  
Joseph T. Wade
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
High Sensitivity ◽  
Purifying Selection ◽  
Ribosome Profiling ◽  
Small Subset ◽  
Stop Codons ◽  
Small Proteins ◽  
Domains Of Life ◽  
Short Orfs

Small proteins of <51 amino acids are abundant across all domains of life but are often overlooked because their small size makes them difficult to predict computationally, and they are refractory to standard proteomic approaches. Ribosome profiling has been used to infer the existence of small proteins by detecting the translation of the corresponding open reading frames (ORFs). Detection of translated short ORFs by ribosome profiling can be improved by treating cells with drugs that stall ribosomes at specific codons. Here, we combine the analysis of ribosome profiling data for Escherichia coli cells treated with antibiotics that stall ribosomes at either start or stop codons. Thus, we identify ribosome-occupied start and stop codons with high sensitivity for ∼400 novel putative ORFs. The newly discovered ORFs are mostly short, with 365 encoding proteins of <51 amino acids. We validate translation of several selected short ORFs, and show that many likely encode unstable proteins. Moreover, we present evidence that most of the newly identified short ORFs are not under purifying selection, suggesting they do not impact cell fitness, although a small subset have the hallmarks of functional ORFs. IMPORTANCE Small proteins of <51 amino acids are abundant across all domains of life but are often overlooked because their small size makes them difficult to predict computationally, and they are refractory to standard proteomic approaches. Recent studies have discovered small proteins by mapping the location of translating ribosomes on RNA using a technique known as ribosome profiling. Discovery of translated sORFs using ribosome profiling can be improved by treating cells with drugs that trap initiating ribosomes. Here, we show that combining these data with equivalent data for cells treated with a drug that stalls terminating ribosomes facilitates the discovery of small proteins. We use this approach to discover 365 putative genes that encode small proteins in Escherichia coli .

scholarly journals Promoter architecture determines co-translational regulation of mRNA

2017 ◽  
Author(s):  
Lorena Espinar ◽  
Miquel Àngel Schikora Tamarit ◽  
Júlia Domingo ◽  
Lucas B. Carey
Keyword(s):  
Gene Expression ◽  
Translational Regulation ◽  
Open Reading Frames ◽  
Mrna Levels ◽  
Rna Seq ◽  
Linear Interaction ◽  
Information Encoding ◽  
Inducible Promoters ◽  
Promoter Architecture ◽  
Inducible Genes

AbstractInformation that regulates gene expression is encoded throughout each gene but if different regulatory regions can be understood in isolation, or if they interact, is unknown. Here we measure mRNA levels for 10,000 open reading frames (ORFs) transcribed from either an inducible or constitutive promoter. We find that the strength of co-translational regulation on mRNA levels is determined by promoter architecture. Using a novel computational-genetic screen of 6402 RNA-seq experiments we identify the RNA helicase Dbp2 as the mechanism by which co-translational regulation is reduced specifically for inducible promoters. Finally, we find that for constitutive genes, but not inducible genes, most of the information encoding regulation of mRNA levels in response to changes in growth rate is encoded in the ORF and not in the promoter. Thus the ORF sequence is a major regulator of gene expression, and a non-linear interaction between promoters and ORFs determines mRNA levels.

scholarly journals Optimal transcriptional regulation of cellular responses to sudden environmental shifts

2020 ◽  
Author(s):  
Daniel Schultz ◽  
Lev S. Tsimring
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Cell Response ◽  
Cellular Responses ◽  
Natural Systems ◽  
Selective Pressures ◽  
Regulatory Strategies ◽  
Regulatory Strategy ◽  
A Cell ◽  
The Many

ABSTRACTCellular responses to sudden changes in their environment require prompt expression of the correct levels of the appropriate enzymes. These enzymes are typically regulated by transcription factors that sense the presence of inducers and control gene expression for the duration of the response. The specific choice of regulatory strategy depends on the characteristics of each cell response, with the pattern of gene expression dictated by parameters such as the affinity of the transcription factor to its binding sites and the strength of the promoters it regulates. Although much is known about how gene regulation determines the dynamics of cell responses, we still lack a framework to understand how the many different regulatory strategies evolved in natural systems relate to the constraints imposed by the selective pressures acting in each particular case. Here, we analyze a dynamical model of a cell response where expression of a transcriptionally repressed enzyme is induced by a sudden exposure to its substrate. We identify strategies of gene regulation that optimize the response for different types of selective pressures, which we define as a set of costs associated with substrate, enzyme and repressor intracellular concentrations during the response. We find that regulated responses happen within a defined region in the parameter space. While responses to costly (toxic) substrates favor the usage of strongly self-regulated repressors, responses where expression of enzyme is more costly than its substrate favor the usage of constitutively expressed repressors. There is only a very narrow range of selective pressures that would favor weakly self-regulated repressors. This framework can be used to infer which costs and benefits are most critical in the evolution of natural examples of cellular responses, and to predict how a response can optimize its regulation when transported to a new environment with different demands.

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