Kinetic competition during the transcription cycle results in stochastic RNA processing

Coulon, Antoine; Ferguson, Matthew L.; Turris, Valeria de; Palangat, Murali; Chow, Carson C.; Larson, Daniel R.

doi:10.7554/elife.03939

Cited by 209 publications

(263 citation statements)

References 63 publications

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“…The previously described construct HIV-2-noGag-BglSL (21) is referred to as 2-noGag-BSL for clarity in the present report; this HIV-2-based construct contains two stop-codon mutations in the gag gene-one at codon 17 of Gag in MA and one at codon 171 of CA-to abolish Gag expression (21). Construct globin-MSL was modified from r-Cfp-betaglobin (27), which encodes a β-globin gene under the control of the tet promoter. The β-globin gene includes introns, and a CeFP gene was fused to the end of exon 3; additionally, intron 2 and 3′ UTR contain stem-loop sequences recognized by bacteriophage PP7 and MS2 coat proteins, respectively.…”

Section: Methodsmentioning

confidence: 99%

HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

Chen

Rahman

Nikolaitchik

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA-Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA-Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.retrovirus | RNA genome | Gag-RNA complex | virus assembly | RNA-binding protein

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Section: Methodsmentioning

confidence: 99%

HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

Chen

Rahman

Nikolaitchik

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Work from the Shav-Tal laboratory (Brody et al 2011) measured the accumulation of snRNPs during splicing and demonstrated that the elongation machinery does not "wait" for each splicing event to go to completion but rather moves ahead so that more introns accumulate. Likewise, adding more introns did not measurably change the rate of transcript synthesis, indicating that elongation proceeds independently of splicing (Brody et al 2011).Further evidence of the relationship between elongation and splicing rates was obtained by Coulon et al (2014). Using dual labeling of intronic and exonic RNA, one is able to separate the kinetics of elongation, splicing, and cleavage.…”

mentioning

confidence: 75%

“…Further evidence of the relationship between elongation and splicing rates was obtained by Coulon et al (2014). Using dual labeling of intronic and exonic RNA, one is able to separate the kinetics of elongation, splicing, and cleavage.…”

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confidence: 86%

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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“…Notably, this allows the simultaneous following of different RNA species in vivo, or it can be used to analyze the kinetics and precise control of mRNA splicing and translation (Martin et al, 2013;Coulon et al, 2014;Halstead et al, 2015).…”

Section: Fluorophore-binding Rna Motifsmentioning

confidence: 99%

Labelling and imaging of single endogenous messenger RNA particlesin vivo

Spille

Kubitscheck

2015

Journal of Cell Science

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RNA molecules carry out widely diverse functions in numerous different physiological processes in living cells. The RNA life cycle from transcription, through the processing of nascent RNA, to the regulatory function of non-coding RNA and cytoplasmic translation of messenger RNA has been studied extensively using biochemical and molecular biology techniques. In this Commentary, we highlight how single molecule imaging and particle tracking can yield further insight into the dynamics of RNA particles in living cells. In the past few years, a variety of bright and photo-stable labelling techniques have been developed to generate sufficient contrast for imaging of single endogenous RNAs in vivo. New imaging modalities allow determination of not only lateral but also axial positions with high precision within the cellular context, and across a wide range of specimen from yeast and bacteria to cultured cells, and even multicellular organisms or live animals. A whole range of methods to locate and track single particles, and to analyze trajectory data are available to yield detailed information about the kinetics of all parts of the RNA life cycle. Although the concepts presented are applicable to all types of RNA, we showcase here the wealth of information gained from in vivo imaging of single particles by discussing studies investigating dynamics of intranuclear trafficking, nuclear pore transport and cytoplasmic transport of endogenous messenger RNA.

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Kinetic competition during the transcription cycle results in stochastic RNA processing

Cited by 209 publications

References 63 publications

HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein

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

Labelling and imaging of single endogenous messenger RNA particlesin vivo

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