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Architecture and RNA binding of the human negative elongation factor

View Article: PubMed Central - PubMed

ABSTRACT

Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF.

Doi:: http://dx.doi.org/10.7554/eLife.14981.001

No MeSH data available.


Related in: MedlinePlus

Overexpression of NELF-A, NELF-B, and NELF-C in 293FT and RNA association.(A) Western blot analysis of NELF expression in 293FT cells in the presence or absence of plasmids overexpressing 3X MYC-NELF-A, 3X FLAG-NELF-C, or 3X FLAG-NELF-B. Cells were treated with 4sU for 16 hr. GAPDH was used as loading control. (B) Immunoprecipitation of 3X MYC-NELF-A, 3X FLAG-NELF-C, or 3X FLAG-NELF-B from 293FT cells by MYC or FLAG specific antibodies, respectively. Lane 1 contains non-transfected cells (NELF-A and -C) or empty vector (NELF-B). Lane 2 contains cells carrying the overexpression plasmid for 3X MYC-NELF-A (upper blot), 3X FLAG-NELF-C (middle blot), or 3X FLAG NELF-B (lower blot). Cells were treated for 16 hr with 4sU prior to UV crosslinking and immunoprecipitation. Western blot analysis was carried out with either MYC or FLAG antibodies. (C) Phosphorimage of SDS PAGE used to resolve 5’ P-32 labeled RNAs crosslinked to overexpressed NELF-A. in 293FT cells. Overexpressed NELF-A was immunoprecipitated by N-terminal 3X MYC tag, (see panel G). Coomassie stained gels of overexpressed NELF-C and -B are shown as a size reference.DOI:http://dx.doi.org/10.7554/eLife.14981.023
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fig8s3: Overexpression of NELF-A, NELF-B, and NELF-C in 293FT and RNA association.(A) Western blot analysis of NELF expression in 293FT cells in the presence or absence of plasmids overexpressing 3X MYC-NELF-A, 3X FLAG-NELF-C, or 3X FLAG-NELF-B. Cells were treated with 4sU for 16 hr. GAPDH was used as loading control. (B) Immunoprecipitation of 3X MYC-NELF-A, 3X FLAG-NELF-C, or 3X FLAG-NELF-B from 293FT cells by MYC or FLAG specific antibodies, respectively. Lane 1 contains non-transfected cells (NELF-A and -C) or empty vector (NELF-B). Lane 2 contains cells carrying the overexpression plasmid for 3X MYC-NELF-A (upper blot), 3X FLAG-NELF-C (middle blot), or 3X FLAG NELF-B (lower blot). Cells were treated for 16 hr with 4sU prior to UV crosslinking and immunoprecipitation. Western blot analysis was carried out with either MYC or FLAG antibodies. (C) Phosphorimage of SDS PAGE used to resolve 5’ P-32 labeled RNAs crosslinked to overexpressed NELF-A. in 293FT cells. Overexpressed NELF-A was immunoprecipitated by N-terminal 3X MYC tag, (see panel G). Coomassie stained gels of overexpressed NELF-C and -B are shown as a size reference.DOI:http://dx.doi.org/10.7554/eLife.14981.023

Mentions: We found that a NELF-E antibody immunoprecipitated the entire NELF complex, as determined by mass spectrometry analysis and Western blotting, in an apparently stoichiometric fashion, allowing us to assess RNA binding by each subunit (Figure 8A and Figure 8—figure supplement 1A–C, Figure 8—figure supplement 2). Bands corresponding to NELF-E and NELF-B/C were readily and reproducibly detected in the radiolabeled sample from both cell lines (Figure 8B and Figure 8—figure supplement 1C). The intensity of the band for NELF-B/C was less than that observed for the NELF-E band, indicating that NELF-B/C may associate more weakly with RNA than NELF-E. This is consistent with the reported high RNA-binding affinity of the NELF-E RRM domain and our biochemical results (Pagano et al., 2014; Rao et al., 2006). Immunoprecipitation with a NELF-A antibody produced similar results in Jurkat cells (Figure 8—figure supplement 1D). To confirm that NELF-C binds RNA, the NELF-A, -B and -C subunits were cloned into mammalian expression vectors and overexpressed in 293FT cells. Consistent with the native protein, the overexpressed NELF-B and NELF-C subunits bound RNA whereas the NELF-A subunit failed to associate with RNA (Figure 8C, Figure 8—figure supplement 3A–C). Together these results indicate that NELF-B, -C, and -E all associate with RNA in cells.10.7554/eLife.14981.020Figure 8.NELF association with RNA in cells.


Architecture and RNA binding of the human negative elongation factor
Overexpression of NELF-A, NELF-B, and NELF-C in 293FT and RNA association.(A) Western blot analysis of NELF expression in 293FT cells in the presence or absence of plasmids overexpressing 3X MYC-NELF-A, 3X FLAG-NELF-C, or 3X FLAG-NELF-B. Cells were treated with 4sU for 16 hr. GAPDH was used as loading control. (B) Immunoprecipitation of 3X MYC-NELF-A, 3X FLAG-NELF-C, or 3X FLAG-NELF-B from 293FT cells by MYC or FLAG specific antibodies, respectively. Lane 1 contains non-transfected cells (NELF-A and -C) or empty vector (NELF-B). Lane 2 contains cells carrying the overexpression plasmid for 3X MYC-NELF-A (upper blot), 3X FLAG-NELF-C (middle blot), or 3X FLAG NELF-B (lower blot). Cells were treated for 16 hr with 4sU prior to UV crosslinking and immunoprecipitation. Western blot analysis was carried out with either MYC or FLAG antibodies. (C) Phosphorimage of SDS PAGE used to resolve 5’ P-32 labeled RNAs crosslinked to overexpressed NELF-A. in 293FT cells. Overexpressed NELF-A was immunoprecipitated by N-terminal 3X MYC tag, (see panel G). Coomassie stained gels of overexpressed NELF-C and -B are shown as a size reference.DOI:http://dx.doi.org/10.7554/eLife.14981.023
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fig8s3: Overexpression of NELF-A, NELF-B, and NELF-C in 293FT and RNA association.(A) Western blot analysis of NELF expression in 293FT cells in the presence or absence of plasmids overexpressing 3X MYC-NELF-A, 3X FLAG-NELF-C, or 3X FLAG-NELF-B. Cells were treated with 4sU for 16 hr. GAPDH was used as loading control. (B) Immunoprecipitation of 3X MYC-NELF-A, 3X FLAG-NELF-C, or 3X FLAG-NELF-B from 293FT cells by MYC or FLAG specific antibodies, respectively. Lane 1 contains non-transfected cells (NELF-A and -C) or empty vector (NELF-B). Lane 2 contains cells carrying the overexpression plasmid for 3X MYC-NELF-A (upper blot), 3X FLAG-NELF-C (middle blot), or 3X FLAG NELF-B (lower blot). Cells were treated for 16 hr with 4sU prior to UV crosslinking and immunoprecipitation. Western blot analysis was carried out with either MYC or FLAG antibodies. (C) Phosphorimage of SDS PAGE used to resolve 5’ P-32 labeled RNAs crosslinked to overexpressed NELF-A. in 293FT cells. Overexpressed NELF-A was immunoprecipitated by N-terminal 3X MYC tag, (see panel G). Coomassie stained gels of overexpressed NELF-C and -B are shown as a size reference.DOI:http://dx.doi.org/10.7554/eLife.14981.023
Mentions: We found that a NELF-E antibody immunoprecipitated the entire NELF complex, as determined by mass spectrometry analysis and Western blotting, in an apparently stoichiometric fashion, allowing us to assess RNA binding by each subunit (Figure 8A and Figure 8—figure supplement 1A–C, Figure 8—figure supplement 2). Bands corresponding to NELF-E and NELF-B/C were readily and reproducibly detected in the radiolabeled sample from both cell lines (Figure 8B and Figure 8—figure supplement 1C). The intensity of the band for NELF-B/C was less than that observed for the NELF-E band, indicating that NELF-B/C may associate more weakly with RNA than NELF-E. This is consistent with the reported high RNA-binding affinity of the NELF-E RRM domain and our biochemical results (Pagano et al., 2014; Rao et al., 2006). Immunoprecipitation with a NELF-A antibody produced similar results in Jurkat cells (Figure 8—figure supplement 1D). To confirm that NELF-C binds RNA, the NELF-A, -B and -C subunits were cloned into mammalian expression vectors and overexpressed in 293FT cells. Consistent with the native protein, the overexpressed NELF-B and NELF-C subunits bound RNA whereas the NELF-A subunit failed to associate with RNA (Figure 8C, Figure 8—figure supplement 3A–C). Together these results indicate that NELF-B, -C, and -E all associate with RNA in cells.10.7554/eLife.14981.020Figure 8.NELF association with RNA in cells.

View Article: PubMed Central - PubMed

ABSTRACT

Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF.

Doi:: http://dx.doi.org/10.7554/eLife.14981.001

No MeSH data available.


Related in: MedlinePlus