Single-Molecule Imaging Reveals a Switch between Spurious and Functional ncRNA Transcription

Lenstra, Tineke L.; Coulon, Antoine; Chow, Carson C.; Larson, Daniel R.

doi:10.1016/j.molcel.2015.09.028

Cited by 117 publications

(140 citation statements)

References 40 publications

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“…Thus, in a genome-wide study, antisense ncRNA transcription was found to switch off corresponding sense genes under conditions of low, but not high, expression (Xu et al 2011). Similarly, a ncRNA transcribed antisense of the GAL10 gene suppressed leaky expression of GAL10 and GAL1 in glucose-containing repressing medium but not in galactose-containing inducing medium (Lenstra et al 2015). The inhibitory effect of the MIR under conditions of both low and high Pol II transcription may be a specificity of an antisense Pol III transcription unit as opposed to an antisense Pol II ncRNA transcription unit, or the range of the EGFP assay may not cover Pol II expression levels that might be differentially affected by MIR expression.…”

Section: Discussionmentioning

confidence: 94%

Transcriptional interference by RNA polymerase III affects expression of the Polr3e gene

et al. 2017

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Overlapping gene arrangements can potentially contribute to gene expression regulation. A mammalian interspersed repeat (MIR) nested in antisense orientation within the first intron of the Polr3e gene, encoding an RNA polymerase III (Pol III) subunit, is conserved in mammals and highly occupied by Pol III. Using a fluorescence assay, CRISPR/Cas9-mediated deletion of the MIR in mouse embryonic stem cells, and chromatin immunoprecipitation assays, we show that the MIR affects Polr3e expression through transcriptional interference. Our study reveals a mechanism by which a Pol II gene can be regulated at the transcription elongation level by transcription of an embedded antisense Pol III gene.

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Section: Discussionmentioning

confidence: 94%

Transcriptional interference by RNA polymerase III affects expression of the Polr3e gene

et al. 2017

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“…In S. cerevisiae, sense/antisense pairing has been debated, as it was first reported that sense and antisense transcripts never coexist in the same cell (Castelnuovo et al 2013). However, recent studies confirmed that although transcribed in a bimodal fashion, sense and antisense RNAs can coexist within the same cell (Nguyen et al 2014;Lenstra et al 2015) and form dsRNA structures in vivo (Drinnenberg et al 2011;Sinturel et al 2015;Wery et al 2016). In S. pombe, the most likely explanation is that Dicer and the asXUT/ mRNA duplexes are prevented for interaction due to different subcellular localization.…”

Section: Discussionmentioning

confidence: 99%

Native elongating transcript sequencing reveals global anti-correlation between sense and antisense nascent transcription in fission yeast

Wéry

Gautier

Descrimes

et al. 2017

RNA

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Antisense transcription can regulate sense gene expression. However, previous annotations of antisense transcription units have been based on detection of mature antisense long noncoding (aslnc)RNAs by RNA-seq and/or microarrays, only giving a partial view of the antisense transcription landscape and incomplete molecular bases for antisense-mediated regulation. Here, we used native elongating transcript sequencing to map genome-wide nascent antisense transcription in fission yeast. Strikingly, antisense transcription was detected for most protein-coding genes, correlating with low sense transcription, especially when overlapping the mRNA start site. RNA profiling revealed that the resulting aslncRNAs mainly correspond to cryptic Xrn1/Exo2-sensitive transcripts (XUTs). ChIP-seq analyses showed that antisense (as)XUT's expression is associated with specific histone modification patterns. Finally, we showed that asXUTs are controlled by the histone chaperone Spt6 and respond to meiosis induction, in both cases anti-correlating with levels of the paired-sense mRNAs, supporting physiological significance to antisense-mediated gene attenuation. Our work highlights that antisense transcription is much more extended than anticipated and might constitute an additional nonpromoter determinant of gene regulation complexity.

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“…These "transcriptional bursts" amount to "memory" between transcripts: The synthesis of one transcript is likely to be accompanied by the synthesis of another over certain time intervals. Transcriptional bursting has now been observed in yeast (Zenklusen et al 2008;Lenstra et al 2015), slime mold (Chubb et al 2006;Muramoto et al 2010;Stevense et al 2010), fly (Garcia et al 2013), mouse , and human (Yunger et al 2010) cell lines and is a significant source of expression variation between cells. In fact, subsequent steps of gene expression such as RNA export, RNA decay, translation, etc.…”

Section: Transcriptional Burstingmentioning

confidence: 99%

“…Work from many laboratories indicates that all quantities appear to be regulated. Burst size ranges from about two RNA per burst (Lenstra et al 2015) to hundreds (Raj et al 2006), and burst frequency ranges from minutes to hours. Some genes (such as housekeeping genes in yeast) do not appear to show bursting at all, and their expression levels are well approximated by a Poisson distribution (Zenklusen et al 2008;Gandhi et al 2011).…”

Section: Transcriptional Burstingmentioning

confidence: 99%

What have single-molecule studies taught us about gene expression?

Chen

Larson

2016

Genes Dev.

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The production of a single mRNA is the result of many sequential steps, from docking of transcription factors to polymerase initiation, elongation, splicing, and, finally, termination. Much of our knowledge about the fundamentals of RNA synthesis and processing come from ensemble in vitro biochemical measurements. Single-molecule approaches are very much in this same reductionist tradition but offer exquisite sensitivity in space and time along with the ability to observe heterogeneous behavior and actually manipulate macromolecules. These techniques can also be applied in vivo, allowing one to address questions in living cells that were previously restricted to reconstituted systems. In this review, we examine the unique insights that single-molecule techniques have yielded on the mechanisms of gene expression.Single-molecule experiments are now pervasive in biology. What started out as an experimental approach for characterizing ion channels in the 1970s (Neher and Sakmann 1976) has now become a fixture in hundreds of laboratories addressing fundamental questions in biochemistry, cell biology, genetics, and development. The methodology is nearly as diverse as the problems that are addressed and encompasses imaging, optical tweezers, atomic force microscopy, electrophysiology, and cryo-electron microscopy (cryo-EM), to list a few. The unifying principle behind these approaches is straightforward: the ability to observe the heterogeneous, rare, or fleeting behavior of macromolecules that is normally masked by ensemble techniques. In this review, we focus on the role that single-molecule approaches have played in advancing our understanding of the early steps in gene expression.In general, transcription by RNA polymerase (RNAP) in bacteria or RNA polymerase II (Pol II) in eukaryotes begins when transcription factors (TFs) are recruited to the promoter. This leads to the assembly of the preinitiation complex (PIC) that contains a semicompetent polymerase. The PIC is necessary for unwinding duplex DNA and setting the stage for processive elongation by the polymerase. After conformational changes in the PIC, the polymerase escapes the promoter region and enters into productive elongation. In eukaryotes, the nascent RNA undergoes further modifications such as addition of a 5 ′ cap, synthesis of a poly-A tail, and splicing before the mature mRNA is formed. These early steps of gene expression are uniquely suited to elucidation through singlemolecule methods. For example, single-molecule experimental approaches allow one to visualize the order of assembly for molecular complexes (i.e., the PIC) and observe the variety of pathways that can result in initiation of RNAP. The ability to observe kinetics in unperturbed systems can provide clues to the mechanisms of transcription. RNAPs can also be manipulated by singlemolecule optical trapping to reveal the inner workings of force generation by this enzyme. Furthermore, singlemolecule imaging has also revealed the heterogeneity of gene expression that exists among cells in ...

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Single-Molecule Imaging Reveals a Switch between Spurious and Functional ncRNA Transcription

Cited by 117 publications

References 40 publications

Transcriptional interference by RNA polymerase III affects expression of the Polr3e gene

Transcriptional interference by RNA polymerase III affects expression of the Polr3e gene

Native elongating transcript sequencing reveals global anti-correlation between sense and antisense nascent transcription in fission yeast

What have single-molecule studies taught us about gene expression?

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