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

The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. Here, we use in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE) that is required for sequence specific RNA recognition (NBE: CUGAGGA(U) for Drosophila). An NBE-like element is present within the loop region of the transactivation-response element (TAR) of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. We also identify a non-conserved region within the RRM that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, we analyzed promoter-proximal regions genome-wide in Drosophila and show that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, we observe a significant increase in NBEs among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes.

[1]  Harold G. Craighead,et al.  Multiplexed Microcolumn-Based Process for Efficient Selection of RNA Aptamers , 2013, Analytical chemistry.

[2]  H. Handa,et al.  Evidence that Negative Elongation Factor Represses Transcription Elongation through Binding to a DRB Sensitivity-Inducing Factor/RNA Polymerase II Complex and RNA , 2002, Molecular and Cellular Biology.

[3]  D. Price,et al.  Control of RNA Polymerase II Elongation Potential by a Novel Carboxyl-terminal Domain Kinase* , 1996, The Journal of Biological Chemistry.

[4]  D. Price,et al.  Purification of P-TEFb, a Transcription Factor Required for the Transition into Productive Elongation (*) , 1995, The Journal of Biological Chemistry.

[5]  H. Handa,et al.  NELF interacts with CBC and participates in 3' end processing of replication-dependent histone mRNAs. , 2007, Molecular cell.

[6]  C. Dominguez,et al.  The RNA recognition motif, a plastic RNA‐binding platform to regulate post‐transcriptional gene expression , 2005, The FEBS journal.

[7]  Charles Elkan,et al.  Fitting a Mixture Model By Expectation Maximization To Discover Motifs In Biopolymer , 1994, ISMB.

[8]  Brian M. Farley,et al.  Molecular Basis of RNA Recognition by the Embryonic Polarity Determinant MEX-5* , 2007, Journal of Biological Chemistry.

[9]  S. Ryder,et al.  Quantitative approaches to monitor protein-nucleic acid interactions using fluorescent probes. , 2011, RNA.

[10]  D. Gilmour,et al.  Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex , 2010, Proceedings of the National Academy of Sciences.

[11]  Leighton J. Core,et al.  Precise Maps of RNA Polymerase Reveal How Promoters Direct Initiation and Pausing , 2013, Science.

[12]  K. Minton,et al.  Screening of λ library for differentially expressed genes using in vitro transcripts , 1985 .

[13]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[14]  D. Marquardt.  An Algorithm for Least-Squares Estimation of Nonlinear Parameters , 1963 .

[15]  Leighton J. Core,et al.  Defining the status of RNA polymerase at promoters. , 2012, Cell reports.

[16]  J. N. Rao,et al.  Structural studies on the RNA-recognition motif of NELF E, a cellular negative transcription elongation factor involved in the regulation of HIV transcription. , 2006, The Biochemical journal.

[17]  Ping Wei,et al.  A Novel CDK9-Associated C-Type Cyclin Interacts Directly with HIV-1 Tat and Mediates Its High-Affinity, Loop-Specific Binding to TAR RNA , 1998, Cell.

[18]  John T. Lis,et al.  Defining mechanisms that regulate RNA polymerase II transcription in vivo , 2009, Nature.

[19]  Leighton J. Core,et al.  Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters , 2008, Science.

[20]  M. Singh,et al.  HIV‐1 tat protein stimulates transcription by binding to a U‐rich bulge in the stem of the TAR RNA structure. , 1990, The EMBO journal.

[21]  Wilfred W. Li,et al.  MEME: discovering and analyzing DNA and protein sequence motifs , 2006, Nucleic Acids Res..

[22]  D. Lilley,et al.  The importance of G.A hydrogen bonding in the metal ion- and protein-induced folding of a kink turn RNA. , 2008, Journal of molecular biology.

[23]  W. Webb,et al.  P-TEFb Is Critical for the Maturation of RNA Polymerase II into Productive Elongation In Vivo , 2007, Molecular and Cellular Biology.

[24]  K. Yano,et al.  DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. , 1998, Genes & development.

[25]  J. Lis,et al.  P-TEFb kinase recruitment and function at heat shock loci. , 2000, Genes & development.

[26]  Jonathan Karn,et al.  Transcriptional and posttranscriptional regulation of HIV-1 gene expression. , 2012, Cold Spring Harbor perspectives in medicine.

[27]  B. Peterlin,et al.  Dynamics of Human Immunodeficiency Virus Transcription: P-TEFb Phosphorylates RD and Dissociates Negative Effectors from the Transactivation Response Element , 2004, Molecular and Cellular Biology.

[28]  Mark D. Biggin,et al.  NELF and GAGA Factor Are Linked to Promoter-Proximal Pausing at Many Genes in Drosophila , 2008, Molecular and Cellular Biology.

[29]  Marcel Martin.  Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .

[30]  Qiang Zhou,et al.  The control of HIV transcription: keeping RNA polymerase II on track. , 2011, Cell host & microbe.

[31]  J. Lis,et al.  Transcription Factor and Polymerase Recruitment, Modification, and Movement on dhsp70 In Vivo in the Minutes following Heat Shock , 2003, Molecular and Cellular Biology.

[32]  J. Coulter.  The Importance of Grass , 1956 .

[33]  Christian Cole,et al.  The Jpred 3 secondary structure prediction server , 2008, Nucleic Acids Res..

[34]  David R. Latulippe,et al.  RAPID-SELEX for RNA Aptamers , 2013, PLoS ONE.

[35]  K. Yano,et al.  Human Transcription Elongation Factor NELF: Identification of Novel Subunits and Reconstitution of the Functionally Active Complex , 2003, Molecular and Cellular Biology.

[36]  G J Barton,et al.  Application of multiple sequence alignment profiles to improve protein secondary structure prediction , 2000, Proteins.

[37]  J. Karn,et al.  Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[38]  Brian M. Farley,et al.  RNA recognition by the embryonic cell fate determinant and germline totipotency factor MEX-3 , 2009, Proceedings of the National Academy of Sciences.

[39]  T. D. Schneider,et al.  Sequence logos: a new way to display consensus sequences. , 1990, Nucleic acids research.

[40]  David Haussler,et al.  The UCSC genome browser database: update 2007 , 2006, Nucleic Acids Res..

[41]  Hiroshi Handa,et al.  NELF, a Multisubunit Complex Containing RD, Cooperates with DSIF to Repress RNA Polymerase II Elongation , 1999, Cell.

[42]  Eric C. Holland,et al.  HIV-1 tat trans-activation requires the loop sequence within tar , 1988, Nature.

[43]  R. Overbeek,et al.  Searching for patterns in genomic data. , 1997, Trends in genetics : TIG.

[44]  J. N. Rao,et al.  NELF-E RRM undergoes major structural changes in flexible protein regions on target RNA binding. , 2008, Biochemistry.

[45]  Leighton J. Core,et al.  Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells. , 2011, Genes & development.

[46]  Z. Derewenda,et al.  Overcoming expression and purification problems of RhoGDI using a family of "parallel" expression vectors. , 1999, Protein expression and purification.

[47]  S. Ryder,et al.  End-labeling oligonucleotides with chemical tags after synthesis. , 2012, Methods in molecular biology.

[48]  Leping Li,et al.  NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly. , 2008, Genes & development.

[49]  D. Fargo,et al.  Global Analysis of Short RNAs Reveals Widespread Promoter-Proximal Stalling and Arrest of Pol II in Drosophila , 2010, Science.

[50]  Charles Y. Lin,et al.  SR Proteins Collaborate with 7SK and Promoter-Associated Nascent RNA to Release Paused Polymerase , 2013, Cell.

[51]  Mary Goldman,et al.  The UCSC Genome Browser database: update 2011 , 2010, Nucleic Acids Res..

[52]  Stephen M. Mount,et al.  The genome sequence of Drosophila melanogaster. , 2000, Science.

[53]  John T. Lis,et al.  Breaking barriers to transcription elongation , 2006, Nature Reviews Molecular Cell Biology.

[54]  Christopher B. Burge,et al.  c-Myc Regulates Transcriptional Pause Release , 2010, Cell.

[55]  Manolis Kellis,et al.  RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo , 2007, Nature Genetics.

[56]  S. Henikoff,et al.  Amino acid substitution matrices from protein blocks. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[57]  R. Mayeux,et al.  Epidemiology of Alzheimer disease. , 2012, Cold Spring Harbor perspectives in medicine.

[58]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[59]  Bin Xie,et al.  Pausing of RNA Polymerase II Disrupts DNA-Specified Nucleosome Organization to Enable Precise Gene Regulation , 2010, Cell.

[60]  T. Steitz,et al.  The kink‐turn: a new RNA secondary structure motif , 2001, The EMBO journal.