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.
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
John M
JM
Pagano
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
Hojoong
H
Kwak
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
Colin T
CT
Waters
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
Rebekka O
RO
Sprouse
Division of Oncology, Department of Internal Medicine, Washington University School of Medicine, St Louis, Missouri, United States of America.
Brian S
BS
White
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
Abdullah
A
Ozer
School of Applied and Engineering Physics, Cornell University, Ithaca, New York, United States of America.
Kylan
K
Szeto
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
David
D
Shalloway
School of Applied and Engineering Physics, Cornell University, Ithaca, New York, United States of America.
Harold G
HG
Craighead
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.
John T
JT
Lis
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics; D015658:HIV Infections / Q000235:genetics; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D059372:Nucleotide Motifs / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
D016601:RNA-Binding Proteins; D014157:Transcription Factors; C118437:negative elongation factor; D012313:RNA; D012319:RNA Polymerase II