Solution structure of the 5′-terminal hairpin of the 7SK small nuclear RNA

Bourbigot, Sarah; Dock-Brégeon, Anne-Catherine; Eberling, Pascal; Coutant, Jérôme; Kieffer, Bruno; Lebars, Isabelle

doi:10.1261/rna.056523.116

Cited by 22 publications

(15 citation statements)

References 88 publications

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“…3A,C). We were also able to observe mild reactivity at U68 which demonstrates flexible base-pairing in molecular dynamics simulations (Martinez-Zapien et al 2017), NMR (Lebars et al 2010;Bourbigot et al 2016), and SHAPE probing (Lebars et al 2010). These together suggest that EDC, as well as high concentrations of CMC, can be used for the probing of RNA conformation in intact cells.…”

Section: Edc Is a Robust Chemical Probe Of Rna Conformation In Cellsmentioning

confidence: 53%

“…The 7SK RNA has a well-documented, conserved secondary structure (Marz et al 2009). The first major stem-loop in 7SK has been extensively studied with in vitro chemical probing (Wassarman and Steitz 1991;Brogie and Price 2017), NMR (Bourbigot et al 2016), and X-ray crystallography (Martinez-Zapien et al 2017), revealing its multiple U-bulges and loops with single-stranded U, thus providing a point of comparison for exposed bases that should be detectable by chemical probing.…”

Section: Edc Is a Robust Chemical Probe Of Rna Conformation In Cellsmentioning

confidence: 99%

“…S4B). In particular, crystallography and NMR identified U40, U41, U63, and U72 to be unpaired and lacking tertiary interactions (Bourbigot et al 2016;Martinez-Zapien et al 2017). These were strongly captured in all of our EDC and 90 mM CMC probing results (Fig.…”

Section: Edc Is a Robust Chemical Probe Of Rna Conformation In Cellsmentioning

confidence: 99%

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Carbodiimide reagents for the chemical probing of RNA structure in cells

Wang

Sexton

Culligan

et al. 2018

RNA

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Deciphering the conformations of RNAs in their cellular environment allows identification of RNA elements with potentially functional roles within biological contexts. Insight into the conformation of RNA in cells has been achieved using chemical probes that were developed to react specifically with flexible RNA nucleotides, or the Watson-Crick face of singlestranded nucleotides. The most widely used probes are either selective SHAPE (2 ′ ′ ′ ′ ′ -hydroxyl acylation and primer extension) reagents that probe nucleotide flexibility, or dimethyl sulfate (DMS), which probes the base-pairing at adenine and cytosine but is unable to interrogate guanine or uracil. The constitutively charged carbodiimide N-cyclohexyl-N ′ ′ ′ ′ ′ -(2morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) is widely used for probing G and U nucleotides, but has not been established for probing RNA in cells. Here, we report the use of a smaller and conditionally charged reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), as a chemical probe of RNA conformation, and the first reagent validated for structure probing of unpaired G and U nucleotides in intact cells. We showed that EDC demonstrates similar reactivity to CMC when probing transcripts in vitro. We found that EDC specifically reacted with accessible nucleotides in the 7SK noncoding RNA in intact cells. We probed structured regions within the Xist lncRNA with EDC and integrated these data with DMS probing data. Together, EDC and DMS allowed us to refine predicted structure models for the 3 ′ ′ ′ ′ ′ extension of repeat C within Xist. These results highlight how complementing DMS probing experiments with EDC allows the analysis of Watson-Crick base-pairing at all four nucleotides of RNAs in their cellular context.

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Section: Edc Is a Robust Chemical Probe Of Rna Conformation In Cellsmentioning

confidence: 53%

Section: Edc Is a Robust Chemical Probe Of Rna Conformation In Cellsmentioning

confidence: 99%

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Carbodiimide reagents for the chemical probing of RNA structure in cells

Wang

Sexton

Culligan

et al. 2018

RNA

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“…The 7SK snRNA is a highly structured RNA that functions to regulate transcription in metazoans (54). Stem I of the 7SK snRNA encompasses the first 108 nucleotides, which are organized as a GC-rich stem-loop (55). We chose this RNA as a surrogate for a nonviral RNA.…”

Section: Figmentioning

confidence: 99%

Reovirus Nonstructural Protein σNS Acts as an RNA Stability Factor Promoting Viral Genome Replication

Zamora

Knowlton

et al. 2018

J Virol

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Viral nonstructural proteins, which are not packaged into virions, are essential for the replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. The reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, the activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered, using and cell-based RNA degradation experiments, that σNS increases the RNA half-life. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases the viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication. Following infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the family encode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different viruses in the family diverge in primary sequence, they are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Using and cell culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the family assort and replicate their genomes.

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“…The 3! stem loop of 7SK has a similar structure including a twonucleotide bulge (Bourbigot et al, 2016;Uchikawa et al, 2015). We hypothesized that a similar interaction of 7SK with the #3 helix of LARP7 might expose the MID.…”

Section: Mepcementioning

confidence: 99%

Reconstitution and functional analysis of the 7SK snRNP

Brogie¹

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ACKNOWLEDGEMENTSFirst and foremost, I would like to thank my wife Ashley for her loving patience during this adventure and my daughters Hazel and Eleanor for giving me the motivation to succeed even during the most challenging times. I would also like to thank my thesis advisor Dr. David Price for providing me with incredible opportunities and challenging me to be the best scientist I can be. He has always championed for my success before and during my graduate career and taught me to appreciate thinking about the details. I would also like to thank the current and past members of the Price lab, specifically Dr. Kyle Nilson providing many hours of scientific and philosophic discussion. Kyle also expanded my understanding about what can be considered music.Finally, I would like to thank my parents, family, and close friends who have provided me with support and understanding during these many years.iii ABSTRACT Every cell in the human body contains almost the same genetic material, therefore, cellular identity is derived from the selection of genes transcribed into RNA and the mRNAs that are made into proteins. To achieve precise control of gene expression, the transcription of messenger RNAs by RNA polymerase II is regulated at multiple checkpoints. A major control point within this system is the P-TEFb dependent transition from paused to productive elongating polymerase complexes. Reversible inhibition of P-TEFb by the 7SK small nuclear ribonucleoprotein (snRNP) is the key step in the control of transcription elongation. Due to the importance of the regulation of P-TEFb, this research investigates the structure of 7SK RNA and the interactions within the 7SK snRNP. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) was used to demonstrate a magnesiumdependent conformational change of in vitro transcribed 7SK RNA folding including a switch in the pairing of the 7SK motif, which is required for P-TEFb regulation. SHAPE was also used to determine that the 5′ end of 7SK pairs alternatively with two different regions within the RNA resulting in open and closed conformations. Moreover, SHAPE was used to show a similar conformational change in cellular 7SK snRNP complexes after the loss of P-TEFb. Assembly of the 7SK snRNP in vitro, using recombinant HEXIM1, P-TEFb, LARP7, MEPCE, and in vitro transcribed 7SK RNA were combined under optimized conditions, resulted in a complete and functional complex. These complexes demonstrated a reversible inhibition of the activity of P-TEFb as well as a similar structure to cellular complexes. LARP7 was found to contain a C-terminal MEPCE interaction domain (MID) that associates with and inhibits MEPCE after binding to the 3′ stem loop of 7SK. The inhibition of MEPCE was determined to be dependent on the overall conformation of 7SK and structural elements of the 3′ stem loop. Use of a highly selective degrader of Brd4, dBET6, also revealed a possible alternative mechanism for Piv TEFb sequestration into the 7SK snRNP. Collectively, these findings aid in the understa...

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Solution structure of the 5′-terminal hairpin of the 7SK small nuclear RNA

Cited by 22 publications

References 88 publications

Carbodiimide reagents for the chemical probing of RNA structure in cells

Carbodiimide reagents for the chemical probing of RNA structure in cells

Reovirus Nonstructural Protein σNS Acts as an RNA Stability Factor Promoting Viral Genome Replication

Reconstitution and functional analysis of the 7SK snRNP

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