Nascent Transcript Sequencing for the Mapping of Promoters in thaliana Mitochondria

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.

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Kinetic Analysis of tRNA-Directed Transcription Antitermination of the Bacillus subtilis glyQS Gene In Vitro

Journal of Bacteriology ◽

10.1128/jb.186.16.5392-5399.2004 ◽

2004 ◽

Vol 186 (16) ◽

pp. 5392-5399 ◽

Cited By ~ 38

Author(s):

Frank J. Grundy ◽

Tina M. Henkin

Keyword(s):

Bacillus Subtilis ◽

Assay System ◽

Nascent Transcript ◽

Vitro Assay ◽

T Box ◽

Transcription Antitermination ◽

Transcriptional Pausing ◽

Transcription Complexes ◽

Kinetics Of

ABSTRACT Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. tRNAGly-dependent antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on antitermination efficiency was analyzed. We found that addition of tRNAGly can promote antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.

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Nascent transcript sequencing visualizes transcription at nucleotide resolution

Nature ◽

10.1038/nature09652 ◽

2011 ◽

Vol 469 (7330) ◽

pp. 368-373 ◽

Cited By ~ 510

Author(s):

L. Stirling Churchman ◽

Jonathan S. Weissman

Keyword(s):

Nascent Transcript ◽

Nucleotide Resolution

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Identification of cis Elements Directing Termination of Yeast Nonpolyadenylated snoRNA Transcripts

Molecular and Cellular Biology ◽

10.1128/mcb.24.14.6241-6252.2004 ◽

2004 ◽

Vol 24 (14) ◽

pp. 6241-6252 ◽

Cited By ~ 106

Author(s):

Kristina L. Carroll ◽

Dennis A. Pradhan ◽

Josh A. Granek ◽

Neil D. Clarke ◽

Jeffry L. Corden

Keyword(s):

Rna Polymerase Ii ◽

Binding Sites ◽

Rna Binding ◽

Rna Binding Proteins ◽

Large Degree ◽

Sequence Motifs ◽

Mobility Shift ◽

Nascent Transcript ◽

Pol Ii ◽

Gel Mobility Shift

ABSTRACT RNA polymerase II (Pol II) termination is triggered by sequences present in the nascent transcript. Termination of pre-mRNA transcription is coupled to recognition of cis-acting sequences that direct cleavage and polyadenylation of the pre-mRNA. Termination of nonpolyadenylated [non-poly(A)] Pol II transcripts in Saccharomyces cerevisiae requires the RNA-binding proteins Nrd1 and Nab3. We have used a mutational strategy to characterize non-poly(A) termination elements downstream of the SNR13 and SNR47 snoRNA genes. This approach detected two common RNA sequence motifs, GUA[AG] and UCUU. The first motif corresponds to the known Nrd1-binding site, which we have verified here by gel mobility shift assays. We also show that Nab3 protein binds specifically to RNA containing the UCUU motif. Taken together, our data suggest that Nrd1 and Nab3 binding sites play a significant role in defining non-poly(A) terminators. As is the case with poly(A) terminators, there is no strong consensus for non-poly(A) terminators, and the arrangement of Nrd1p and Nab3p binding sites varies considerably. In addition, the organization of these sequences is not strongly conserved among even closely related yeasts. This indicates a large degree of genetic variability. Despite this variability, we were able to use a computational model to show that the binding sites for Nrd1 and Nab3 can identify genes for which transcription termination is mediated by these proteins.

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Gene expression amplification by nuclear speckle association

The Journal of Cell Biology ◽

10.1083/jcb.201904046 ◽

2019 ◽

pp. jcb.201904046 ◽

Cited By ~ 7

Author(s):

Jiah Kim ◽

Neha Chivukula Venkata ◽

Gabriela Andrea Hernandez Gonzalez ◽

Nimish Khanna ◽

Andrew S. Belmont

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Liquid Droplet ◽

Transcript Level ◽

Rna Degradation ◽

Nuclear Speckles ◽

Nascent Transcript ◽

Nuclear Compartment ◽

Transcript Levels ◽

Endogenous Genes

Many active genes reproducibly position near nuclear speckles, but the functional significance of this positioning is unknown. Here we show that HSPA1B BAC transgenes and endogenous Hsp70 genes turn on 2–4 min after heat shock (HS), irrespective of their distance to speckles. However, both total HSPA1B mRNA counts and nascent transcript levels measured adjacent to the transgene are approximately twofold higher for speckle-associated alleles 15 min after HS. Nascent transcript level fold-increases for speckle-associated alleles are 12–56-fold and 3–7-fold higher 1–2 h after HS for HSPA1B transgenes and endogenous genes, respectively. Severalfold higher nascent transcript levels for several Hsp70 flanking genes also correlate with speckle association at 37°C. Live-cell imaging reveals that HSPA1B nascent transcript levels increase/decrease with speckle association/disassociation. Initial investigation reveals that increased nascent transcript levels accompanying speckle association correlate with reduced exosome RNA degradation and larger Ser2p CTD-modified RNA polymerase II foci. Our results demonstrate stochastic gene expression dependent on positioning relative to a liquid-droplet nuclear compartment through “gene expression amplification.”

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Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis

Journal of Virology ◽

10.1128/jvi.03216-15 ◽

2016 ◽

Vol 90 (11) ◽

pp. 5384-5398 ◽

Cited By ~ 24

Author(s):

Long Liu ◽

Jiao Tian ◽

Hao Nan ◽

Mengmeng Tian ◽

Yuan Li ◽

...

Keyword(s):

Rna Synthesis ◽

Nonstructural Protein ◽

Critical Role ◽

Continuous Extension ◽

Rna Helicase ◽

Viral Rna ◽

Respiratory Syndrome Virus ◽

Nascent Transcript ◽

Multifunctional Protein ◽

N Protein

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis.IMPORTANCEIt is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of longer viral sgmRNAs and gRNA. Our data here provide some new insights into the discontinuous to continuous extension of PRRSV RNA synthesis and also offer a new potential anti-PRRSV strategy targeting the N-Nsp9 and/or N-DHX9 interaction.

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The DNA replication fork can pass RNA polymerase without displacing the nascent transcript

Nature ◽

10.1038/366033a0 ◽

1993 ◽

Vol 366 (6450) ◽

pp. 33-39 ◽

Cited By ~ 66

Author(s):

Bin Liu ◽

Mei Lie Wong ◽

Rachel L. Tinker ◽

E. Peter Geiduschek ◽

Bruce M. Alberts

Keyword(s):

Dna Replication ◽

Rna Polymerase ◽

Replication Fork ◽

Nascent Transcript

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Modular Organization of the NusA- and NusG-Stimulated RNA Polymerase Pause Signal That Participates in the Bacillus subtilis trp Operon Attenuation Mechanism

Journal of Bacteriology ◽

10.1128/jb.00223-17 ◽

2017 ◽

Vol 199 (14) ◽

Cited By ~ 8

Author(s):

Smarajit Mondal ◽

Alexander V. Yakhnin ◽

Paul Babitzke

Keyword(s):

Bacillus Subtilis ◽

Rna Polymerase ◽

Untranslated Region ◽

Modular Organization ◽

Additional Time ◽

Nascent Transcript ◽

Content Type ◽

Attenuation Mechanism ◽

Transcription Attenuation ◽

Rna Hairpin

ABSTRACT The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to the nascent transcript and blocks the formation of an antiterminator structure such that the formation of an overlapping intrinsic terminator causes termination in the 5′ untranslated region (5′ UTR). In the absence of bound TRAP, the antiterminator forms and transcription continues into the trp genes. RNA polymerase pauses at positions U107 and U144 in the 5′ UTR. The general transcription elongation factors NusA and NusG stimulate pausing at both positions. NusG-stimulated pausing at U144 requires sequence-specific contacts with a T tract in the nontemplate DNA (ntDNA) strand within the paused transcription bubble. Pausing at U144 participates in a trpE translation repression mechanism. Since U107 just precedes the critical overlap between the antiterminator and terminator structures, pausing at this position is thought to participate in attenuation. Here we carried out in vitro pausing and termination experiments to identify components of the U107 pause signal and to determine whether pausing affects the termination efficiency in the 5′ UTR. We determined that the U107 and U144 pause signals are organized in a modular fashion containing distinct RNA hairpin, U-tract, and T-tract components. NusA-stimulated pausing was affected by hairpin strength and the U-tract sequence, whereas NusG-stimulated pausing was affected by hairpin strength and the T-tract sequence. We also determined that pausing at U107 results in increased TRAP-dependent termination in the 5′ UTR, implying that NusA- and NusG-stimulated pausing participates in the trp operon attenuation mechanism by providing additional time for TRAP binding. IMPORTANCE The expression of several bacterial operons is controlled by regulated termination in the 5′ untranslated region (5′ UTR). Transcription attenuation is defined as situations in which the binding of a regulatory molecule promotes transcription termination in the 5′ UTR, with the default being transcription readthrough into the downstream genes. RNA polymerase pausing is thought to participate in several attenuation mechanisms by synchronizing the position of RNA polymerase with RNA folding and/or regulatory factor binding, although this has only been shown in a few instances. We found that NusA- and NusG-stimulated pausing participates in the attenuation mechanism controlling the expression of the Bacillus subtilis trp operon by increasing the TRAP-dependent termination efficiency. The pause signal is organized in a modular fashion containing RNA hairpin, U-tract, and T-tract components.

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