Defining NELF-E RNA Binding in HIV-1 and Promoter-Proximal Pause Regions

Pagano, John M.; Kwak, Ho Young; Waters, Colin T.; Sprouse, Rebekka O.; White, Brian S.; Ozer, Abdullah; Szeto, Kylan; Shalloway, David; Craighead, Harold G.; Lis, John T.

doi:10.1371/journal.pgen.1004090

Cited by 61 publications

(78 citation statements)

References 56 publications

(90 reference statements)

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“…This analysis can also be used to identify a minimal aptamer: in fact, HiTS-RAP shows that the sequence outside of the 35 nt minimal aptamer defined in the original publication 17 does not influence binding appreciably (Fig. 4b).…”

Section: Discussionmentioning

confidence: 99%

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing–RNA affinity profiling

et al. 2014

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RNA-protein interactions have critical roles in gene regulation. However, high-throughput methods to quantitatively analyze these interactions are lacking. We adapted an Illumina GAIIx sequencer to make several million such measurements with a High-Throughput Sequencing – RNA Affinity Profiling (HiTS-RAP) assay. Millions of cDNAs are sequenced, bound by the E. coli replication terminator protein Tus, and transcribed in situ, whereupon Tus halts transcription leaving RNA stably attached to its template DNA. The binding of fluorescently-labeled protein is then quantified in the sequencer. We used HiTS-RAP to measure the affinity of mutagenized libraries of GFP-binding and NELF-E binding aptamers to their respective targets and thereby identified regions in both aptamers that are critical for their RNA-protein interaction. We show that mutations additively affect binding affinity of the NELF-E binding aptamer, whose interaction depends mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depends primarily on secondary structure.

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Section: Discussionmentioning

confidence: 99%

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing–RNA affinity profiling

et al. 2014

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“…FP assay was performed as previously described 29 . In vitro transcribed 5S and 5.8S rRNAs were PAGE purified and eluted into 10 mM Tris pH 7.5 buffer.…”

Section: Methodsmentioning

confidence: 99%

SIRT7 Is an RNA-Activated Protein Lysine Deacylase

et al. 2016

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Mammalian SIRT7 is a member of the sirtuin family that regulate multiple biological processes including genome stability, metabolic pathways, stress responses, and tumorigenesis. SIRT7 has been shown to be important for ribosome biogenesis and transcriptional regulation. SIRT7 knockout mice exhibit complications associated with fatty liver and increased aging in hematopoietic stem cells. However, the molecular basis for its biological function remains unclear, in part due to the lack of efficient enzymatic activity in vitro. Previously we have demonstrated that double-stranded DNA could activate SIRT7’s deacetylase activity in vitro, allowing it to deacetylate H3K18 in the context of chromatin. Here we show that RNA can increase the catalytic efficiency of SIRT7 even better and that SIRT7 can remove long chain fatty acyl groups more efficiently than removing acetyl groups. Truncation and mutagenesis studies revealed residues at both the amino and carboxyl termini of SIRT7 that are involved in RNA-binding and important for activity. RNA immunoprecipitation-sequencing (RIP-seq) identified ribosomal RNA (rRNA) as the predominant RNA binding partners of SIRT7. The associated RNA was able to effectively activate the deacetylase and defatty-acylase activities of SIRT7. Knockdown of SIRT7 increased the lysine fatty acylation of several nuclear proteins based on metabolic labeling with an alkyne-tagged fatty acid analog, supporting that the defatty-acylase activity of SIRT7 is physiologically relevant. These findings provide important insights into the biological functions of SIRT7, as well as an improved platform to develop SIRT7 modulators.

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“…These ~60 nt RNAs accumulate in vivo prior to full HIV-1 activation [120]. Interestingly, NELF-E, the RRM-containing, RNA-binding component of NELF [121] that aids HIV-1 leader pausing [122] interacts with the loop of TAR sequence-specifically [123]. Thus, NELF-E may be a chaperone of nascent RNA structure formation for RNAPII, possibly analogous to the function of NusA for bacterial RNAP.…”

Section: Transcription-guided Folding Of Regulatory Nascent Rnasmentioning

confidence: 99%

A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure

Zhang

Landick

2016

Trends in Biochemical Sciences

121

124

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The vectorial (5′-to-3′ at varying velocity) synthesis of RNA by cellular RNA polymerases creates a rugged kinetic landscape, demarcated by frequent, sometimes long-lived pauses. In addition to myriad gene-regulatory roles, these pauses temporally and spatially program the co-transcriptional, hierarchical folding of biologically active RNAs. Conversely, these RNA structures, which form inside or near the RNA exit channel, interact with the polymerase and adjacent protein factors to influence RNA synthesis by modulating pausing, termination, antitermination, and slippage. Here we review the evolutionary origin, mechanistic underpinnings, and regulatory consequences of this interplay between RNA polymerase and nascent RNA structure. We categorize and attempt to rationalize the extensive linkage between the transcriptional machinery and its product, and provide a framework for future studies.

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Defining NELF-E RNA Binding in HIV-1 and Promoter-Proximal Pause Regions

Cited by 61 publications

References 56 publications

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing–RNA affinity profiling

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing–RNA affinity profiling

SIRT7 Is an RNA-Activated Protein Lysine Deacylase

A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure

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