scholarly journals Non-coding AUG circRNAs constitute an abundant and conserved subclass of circles

2018 ◽  
Author(s):  
Lotte VW Stagsted ◽  
Katrine M Nielsen ◽  
Iben Daugaard ◽  
Thomas B Hansen
Keyword(s):  
Species Conservation ◽  
Ribosome Profiling ◽  
Start Codon ◽  
Circular Rnas ◽  
Protein Coding ◽  
Species Analysis ◽  
Bona Fide ◽  
Translational Start Codon ◽  
Comprehensive Classification ◽  
Translational Start

AbstractCircular RNAs (circRNAs) are a subset of non-coding RNAs (ncRNAs) previously considered as products of missplicing. Now, circRNAs are considered functional molecules, although to date, only few functions have been experimentally validated, and therefore the vast majority of circRNAs are without known relevance. Here, based on RNA sequencing from the ENCODE consortium, we identify and characterize a subset of circRNAs, coined AUG circRNAs, encompassing the annotated translational start codon from the protein-coding host genes. AUG circRNAs are more abundantly expressed and conserved than other groups of circRNAs, and they display an Alu-independent mechanism of biogenesis. The AUG circRNAs contain part of bona fide ORF, and in the recent years, several studies have reported cases of circRNA translation. However, using thorough cross-species analysis, extensive ribosome profiling, proteomics analyses, and experimental data on a selected panel of AUG circRNAs, we observe no indications of translation of AUG circRNAs or any other circRNAs. Our data provide a comprehensive classification of circRNAs and, collectively, the data suggest that the AUG circRNAs constitute an abundant subclass of circRNAs produced independently of primate-specific Alu elements. Moreover, AUG circRNAs exhibit high cross-species conservation and are therefore likely to be functionally relevant.

scholarly journals Noncoding AUG circRNAs constitute an abundant and conserved subclass of circles

2019 ◽  
Vol 2 (3) ◽  
pp. e201900398 ◽  
Author(s):  
Lotte VW Stagsted ◽  
Katrine M Nielsen ◽  
Iben Daugaard ◽  
Thomas B Hansen
Keyword(s):  
Ribosome Profiling ◽  
Start Codon ◽  
Circular Rnas ◽  
Protein Coding ◽  
Reading Frame ◽  
Flanking Sequences ◽  
Bona Fide ◽  
Translational Start Codon ◽  
Comprehensive Classification ◽  
Translational Start

Circular RNAs (circRNAs) are a subset of noncoding RNAs previously considered as products of missplicing. Now, circRNAs are considered functional molecules, although to date, only few functions have been experimentally validated. Here, based on RNA sequencing from the ENCODE consortium, we identify and characterize a subset of circRNAs, coined AUG circRNAs, encompassing the annotated translational start codon from the protein-coding host genes. AUG circRNAs are more abundantly expressed and conserved than other groups of circRNAs, and they display flanking sequences that suggest an Alu-independent mechanism of biogenesis. The AUG circRNAs contain part of bona fide open reading frame, and in the recent years, several studies have reported cases of circRNA translation. However, using thorough cross-species analysis, extensive ribosome profiling, proteomics analyses, and experimental data on a selected panel of AUG circRNAs, we observe no indications of translation of AUG circRNAs or any other circRNAs. Our data provide a comprehensive classification of circRNAs and, collectively, the data suggest that the AUG circRNAs constitute an abundant subclass of circRNAs produced independently of primate-specific Alu elements.

scholarly journals Single-molecule FRET studies on the cotranscriptional folding of a thiamine pyrophosphate riboswitch

2017 ◽  
Vol 115 (2) ◽  
pp. 331-336 ◽  
Author(s):  
Heesoo Uhm ◽  
Wooyoung Kang ◽  
Kook Sun Ha ◽  
Changwon Kang ◽  
Sungchul Hohng
Keyword(s):  
Single Molecule ◽  
Start Codon ◽  
Thiamine Pyrophosphate ◽  
Controlled Study ◽  
Fluorescence Studies ◽  
Physical Mechanisms ◽  
Single Molecule Fret ◽  
Transcriptional Pausing ◽  
Translational Start Codon ◽  
Translational Start

Because RNAs fold as they are being synthesized, their transcription rate can affect their folding. Here, we report the results of single-molecule fluorescence studies that characterize the ligand-dependent cotranscriptional folding of the Escherichia coli thiM riboswitch that regulates translation. We found that the riboswitch aptamer folds into the “off” conformation independent of its ligand, but switches to the “on” conformation during transcriptional pausing near the translational start codon. Ligand binding maintains the riboswitch in the off conformation during transcriptional pauses. We expect our assay will permit the controlled study of the two main physical mechanisms that regulate cotranscriptional folding: transcriptional pausing and transcriptional speed.

scholarly journals Quorum Sensing and Iron-Dependent Coordinated Control of Autoinducer-2 Production via Small RNA RyhB in Vibrio vulnificus

2021 ◽  
Author(s):  
Keun-Woo Lee ◽  
Yancheng Wen ◽  
Na-Young Park ◽  
Kun-Soo Kim
Keyword(s):  
Quorum Sensing ◽  
Small Rna ◽  
Vibrio Vulnificus ◽  
Iron Complex ◽  
Start Codon ◽  
Signal Pathways ◽  
Dependent Manner ◽  
Autoinducer 2 ◽  
Translational Start Codon ◽  
Translational Start

Abstract Roles for the non-coding small RNA RyhB in quorum-sensing and iron-dependent gene modulation in the human pathogen V. vulnificus were assessed in this study. Both the quorum sensing master regulator SmcR and the Fur-iron complex were observed to bind to the region upstream of the non-coding small RNA RyhB gene to repress expression, which suggests that RyhB is associated with both quorum-sensing and iron-dependent signaling in this pathogen. We found that expression of LuxS, which is responsible for the biosynthesis of autoinducer-2 (AI-2), was higher in wild type than in a ryhB-deletion isotype. RyhB binds directly to the 5'-UTR of the luxS transcript to form a heteroduplex, which not only stabilizes LuxS mRNA but also disrupts the secondary structure that normally obscures the translational start codon and thereby allows translation of LuxS to begin. The binding of RyhB to LuxS mRNA requires the chaperone protein Hfq, which stabilizes RyhB. These results demonstrate that the small RNA RyhB is a key element associated with feedback control of AI-2 production, and that it inhibits quorum-sensing signaling in an iron-dependent manner. This study, taken together with previous studies, shows that iron availability and cell density signals are funneled to SmcR and RyhB, and that these regulators coordinate cognate signal pathways that result in the proper balance of protein expression in response to environmental conditions.

scholarly journals Characterization of Spodoptera exigua multicapsid nucleopolyhedrovirus ORF17/18, a homologue of Xestia c-nigrum granulovirus ORF129

2002 ◽  
Vol 83 (11) ◽  
pp. 2857-2867 ◽  
Author(s):  
Wilfred F. J. IJkel ◽  
Els C. Roode ◽  
Rob W. Goldbach ◽  
Just M. Vlak ◽  
Douwe Zuidema
Keyword(s):  
Fusion Protein ◽  
Spodoptera Exigua ◽  
Polyclonal Antiserum ◽  
Start Codon ◽  
E Coli ◽  
Gfp Fusion Protein ◽  
Translational Start Codon ◽  
Translational Start ◽  
Race Analysis

Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) contains a number of genes with a homologue found so far only in a distantly related baculovirus. One of these, SeMNPV ORF17/18 (Se17/18) shares 55% amino acid similarity to ORF129 of Xestia c-nigrum granulovirus (XcGV). Se17/18 was transcribed in cultured S. exigua 301 cells, as a polyadenylated transcript of 1·1 kb. 5′-RACE analysis demonstrated that Se17/18 transcripts started at 134, 131 and 126 nt upstream of the putative translational start codon. These sites overlap with a baculovirus consensus early promoter motif. Se17/18 transcripts were detected by Northern blot analysis and RT–PCR with increasing abundance from 8 h to 24 h post infection (p.i.) and still present until 72 h p.i. A C-terminal GFP-fusion protein of Se17/18 was primarily localized in the cytoplasm of Se301 and Sf21 cells. A chicken polyclonal antiserum was raised that reacted specifically to Se17/18 protein produced in E. coli. However, no immunoreactive protein was detected in SeMNPV-infected Se301 cells and S. exigua larvae, neither in concentrated BV and ODV preparations. These observations and the inability to detect a C-terminal GFP-fusion protein of Se17/18 in Se301 cells using a GFP antibody suggest that Se17/18 protein is present, if at all, in spurious amounts. Based on the low homology of the Se17/18 protein to (methyl) transferases its possible involvement in transcription regulation is discussed.

scholarly journals An A-Factor-Dependent Extracytoplasmic Function Sigma Factor (ςAdsA) That Is Essential for Morphological Development in Streptomyces griseus

2000 ◽  
Vol 182 (16) ◽  
pp. 4596-4605 ◽  
Author(s):  
Haruka Yamazaki ◽  
Yasuo Ohnishi ◽  
Sueharu Horinouchi
Keyword(s):  
Sigma Factor ◽  
Streptomyces Griseus ◽  
Transcriptional Activator ◽  
Start Codon ◽  
Morphological Development ◽  
Aerial Hyphae ◽  
Transcriptional Start Point ◽  
Translational Start Codon ◽  
Translational Start ◽  
Nuclease Mapping

ABSTRACT A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) at an extremely low concentration triggers streptomycin production and aerial mycelium formation in Streptomyces griseus. A-factor induces the expression of an A-factor-dependent transcriptional activator, AdpA, essential for both morphological and physiological differentiation by binding to the A-factor receptor protein ArpA, which has bound and repressed the adpA promoter, and dissociating it from the promoter. Nine DNA fragments that were specifically recognized and bound by histidine-tagged AdpA were isolated by cycles of a gel mobility shift-PCR method. One of them was located in front of a gene encoding an extracytoplasmic function ς factor belonging to a subgroup of the primary ς70 family. The cloned gene was named AdpA-dependent sigma factor gene (adsA), and the gene product was named ςAdsA. Transcription ofadsA depended on A-factor and AdpA, since adsAwas transcribed at a very low and constant level in an A-factor-deficient mutant strain or in an adpA-disrupted strain. Consistent with this, transcription of adsA was greatly enhanced at or near the timing of aerial hyphae formation, as determined by low-resolution S1 nuclease mapping. High-resolution S1 mapping determined the transcriptional start point 82 nucleotides upstream of the translational start codon. DNase I footprinting showed that AdpA bound both strands symmetrically between the transcriptional start point and the translational start codon; AdpA protected the antisense strand from positions +7 to +41 with respect to the transcriptional start point and the sense strand from positions +12 to +46. A weak palindrome was found in the AdpA-binding site. The unusual position bound by AdpA as a transcriptional activator, in relation to the promoter, suggested the presence of a mechanism by which AdpA activates transcription of adsA in some unknown way. Disruption of the chromosomal adsA gene resulted in loss of aerial hyphae formation but not streptomycin or yellow pigment production, indicating that ςAdsA is involved only in morphological development and not in secondary metabolic function. The presence of a single copy in each of the Streptomycesspecies examined by Southern hybridization suggests a common role in morphogenesis in this genus.

scholarly journals A Coding Sequence-Embedded Principle Governs Translational Reading Frame Fidelity

Research ◽  
2018 ◽  
Vol 2018 ◽  
pp. 1-15 ◽  
Author(s):  
Ji Wan ◽  
Xiangwei Gao ◽  
Yuanhui Mao ◽  
Xingqian Zhang ◽  
Shu-Bing Qian
Keyword(s):  
18S Rrna ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Start Codon ◽  
Data Sets ◽  
Protein Coding ◽  
Reading Frame ◽  
Codon Composition ◽  
Sequence Elements ◽  
Correct Reading

Upon initiation at a start codon, the ribosome must maintain the correct reading frame for hundreds of codons in order to produce functional proteins. While some sequence elements are able to trigger programmed ribosomal frameshifting (PRF), very little is known about how the ribosome normally prevents spontaneous frameshift errors that can have dire consequences if uncorrected. Using high resolution ribosome profiling data sets, we discovered that the translating ribosome uses the 3′ end of 18S rRNA to scan the AUG-like codons after the decoding process. The postdecoding mRNA:rRNA interaction not only contributes to predominant translational pausing, but also provides a retrospective mechanism to safeguard the ribosome in the correct reading frame. Partially eliminating the AUG-like “sticky” codons in the reporter message leads to increased +1 frameshift errors. Remarkably, mutating the highly conserved CAU triplet of 18S rRNA globally changes the codon “stickiness”. Further supporting the role of “sticky” sequences in reading frame maintenance, the codon composition of open reading frames is highly optimized across eukaryotic genomes. These results suggest an important layer of information embedded within the protein-coding sequences that instructs the ribosome to ensure reading frame fidelity during translation.

scholarly journals The Enterococcus faecalis EbpA Pilus Protein: Attenuation of Expression, Biofilm Formation, and Adherence to Fibrinogen Start with the Rare Initiation Codon ATT

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Maria Camila Montealegre ◽  
Sabina Leanti La Rosa ◽  
Jung Hyeob Roh ◽  
Barrett R. Harvey ◽  
Barbara E. Murray
Keyword(s):  
Protein Synthesis ◽  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Surface Display ◽  
Hospital Setting ◽  
Start Codon ◽  
Initiation Codon ◽  
Content Type ◽  
Translational Start Codon ◽  
Translational Start

ABSTRACTThe endocarditis and biofilm-associated pili (Ebp) are important inEnterococcus faecalispathogenesis, and the pilus tip, EbpA, has been shown to play a major role in pilus biogenesis, biofilm formation, and experimental infections. Based onin silicoanalyses, we previously predicted that ATT is the EbpA translational start codon, not the ATG codon, 120 bp downstream of ATT, which is annotated as the translational start. ATT is rarely used to initiate protein synthesis, leading to our hypothesis that this codon participates in translational regulation of Ebp production. To investigate this possibility, site-directed mutagenesis was used to introduce consecutive stop codons in place of two lysines at positions 5 and 6 from the ATT, to replace the ATT codonin situwith ATG, and then to revert this ATG to ATT; translational fusions ofebpAtolacZwere also constructed to investigate the effect of these start codons on translation. Our results showed that the annotated ATG does not start translation of EbpA, implicating ATT as the start codon; moreover, the presence of ATT, compared to the engineered ATG, resulted in significantly decreased EbpA surface display, attenuated biofilm, and reduced adherence to fibrinogen. Corroborating these findings, the translational fusion with the native ATT as the initiation codon showed significantly decreased expression of β-galactosidase compared to the construct with ATG in place of ATT. Thus, these results demonstrate that the rare initiation codon of EbpA negatively regulates EbpA surface display and negatively affects Ebp-associated functions, including biofilm and adherence to fibrinogen.IMPORTANCEEnterococcus faecalisis among the leading causes of serious infections in the hospital setting, and the endocarditis and biofilm-associated pili (Ebp) have been shown to play significant roles inE. faecalispathogenesis. Understanding the regulation of virulence is important for the development of new approaches to counteract multidrug-resistant pathogens. We previously predicted that ATT, which has been reported to start protein synthesis only in rare instances, is the most likely translational start codon of EbpA inE. faecalis. Here, we demonstrate that ATT is the initiation codon of EbpA and, relative to a constructed ATG start codon, results in smaller amounts of EbpA on the surface of the cells, attenuating biofilm formation and fibrinogen adherence, phenotypes associated with the ability ofE. faecalisto cause infections. This provides the first example of pilus regulation through the use of an ATT initiation codon.

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