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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.


Multiple sequence alignment of the N-terminal region of NELF-C.The alignment compares full-length NELF-C from Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster, Dictyostelium discoideum, Loa loa, Lottia gigantea, and Chlorella variabilis. Residues are colored according to percent conservation with darker shades of blue representing higher conservation. Barrels above the alignment represent α-helices, arrows β-sheets and are colored according to Figure 1B. N- and C-terminal borders of solved crystal structure are indicated. Sequence alignment was done with Mafft (Katoh and Standley, 2013) followed by manual editing and rendered with JALVIEW (Waterhouse et al., 2009). Surprisingly, a part of the NELF-AC complex also exists in the single cell slime mold Dictyostelium discoideum and the green algae Chlorella variabilis. The hypothetical Dictyostelium proteins DDB_G0286295 and DDB_G0268678 share sequence similarity with the crystallized N-terminus of NELF-A (28% identity, 50% similarity) and C-terminal region of NELF-C (33% identity, 53% similarity) (Figure 1B). Similarly, the hypothetical C. variabilis proteins E1ZMT9 and E1Z2I7 also share sequence similarity with the crystallized N-terminus of NELF-A (25% identity, 44% similarity) and C-terminal region of NELF-C (23% identity, 39% similarity). The conservation of many residues in the hydrophobic core and heterodimer interface indicates that the NELF-AC subcomplex exists in this single cell organism. A putative Dictyostelium homolog is also found for a region of human NELF-B comprising residues 1–410 (DDB_G0284195) (Chang et al., 2012). NELF is likely present in both single and multicellular organisms. Plants may also lack NELF, indicating that they may be devoid of promoter proximal pausing or use other mechanisms to regulate gene expression at the level of elongation. Future work would benefit from understanding how NELF evolved and why some organisms either lost or lack the complex.DOI:http://dx.doi.org/10.7554/eLife.14981.005
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fig1s3: Multiple sequence alignment of the N-terminal region of NELF-C.The alignment compares full-length NELF-C from Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster, Dictyostelium discoideum, Loa loa, Lottia gigantea, and Chlorella variabilis. Residues are colored according to percent conservation with darker shades of blue representing higher conservation. Barrels above the alignment represent α-helices, arrows β-sheets and are colored according to Figure 1B. N- and C-terminal borders of solved crystal structure are indicated. Sequence alignment was done with Mafft (Katoh and Standley, 2013) followed by manual editing and rendered with JALVIEW (Waterhouse et al., 2009). Surprisingly, a part of the NELF-AC complex also exists in the single cell slime mold Dictyostelium discoideum and the green algae Chlorella variabilis. The hypothetical Dictyostelium proteins DDB_G0286295 and DDB_G0268678 share sequence similarity with the crystallized N-terminus of NELF-A (28% identity, 50% similarity) and C-terminal region of NELF-C (33% identity, 53% similarity) (Figure 1B). Similarly, the hypothetical C. variabilis proteins E1ZMT9 and E1Z2I7 also share sequence similarity with the crystallized N-terminus of NELF-A (25% identity, 44% similarity) and C-terminal region of NELF-C (23% identity, 39% similarity). The conservation of many residues in the hydrophobic core and heterodimer interface indicates that the NELF-AC subcomplex exists in this single cell organism. A putative Dictyostelium homolog is also found for a region of human NELF-B comprising residues 1–410 (DDB_G0284195) (Chang et al., 2012). NELF is likely present in both single and multicellular organisms. Plants may also lack NELF, indicating that they may be devoid of promoter proximal pausing or use other mechanisms to regulate gene expression at the level of elongation. Future work would benefit from understanding how NELF evolved and why some organisms either lost or lack the complex.DOI:http://dx.doi.org/10.7554/eLife.14981.005

Mentions: The crystallized regions of human NELF-AC share considerable homology among metazoans, particularly at residues forming the hydrophobic cores and the interface between NELF-A and NELF-C (Figure 1B). The extent of conservation is evident when human and Drosophila melanogaster are compared, which share 55% identity for NELF-A and 50% identity for NELF-C. Intriguingly, NELF-A and -C homologs are present in some worms such as the filiarial nematode Loa loa (Figure 1—figure supplements 2, 3) and single celled organisms such as the green algae Chlorella variabilis and the slime mold Dictyostelium discoideum (Figure 1—figure supplements 2, 3). Most regions outside of the crystallized NELF-AC core diverge between single celled organisms and metazoans (Figure 1—figure supplements 2, 3). Such conservation suggests that NELF may have been present in early eukaryotes and was lost in certain lineages over time.


Architecture and RNA binding of the human negative elongation factor
Multiple sequence alignment of the N-terminal region of NELF-C.The alignment compares full-length NELF-C from Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster, Dictyostelium discoideum, Loa loa, Lottia gigantea, and Chlorella variabilis. Residues are colored according to percent conservation with darker shades of blue representing higher conservation. Barrels above the alignment represent α-helices, arrows β-sheets and are colored according to Figure 1B. N- and C-terminal borders of solved crystal structure are indicated. Sequence alignment was done with Mafft (Katoh and Standley, 2013) followed by manual editing and rendered with JALVIEW (Waterhouse et al., 2009). Surprisingly, a part of the NELF-AC complex also exists in the single cell slime mold Dictyostelium discoideum and the green algae Chlorella variabilis. The hypothetical Dictyostelium proteins DDB_G0286295 and DDB_G0268678 share sequence similarity with the crystallized N-terminus of NELF-A (28% identity, 50% similarity) and C-terminal region of NELF-C (33% identity, 53% similarity) (Figure 1B). Similarly, the hypothetical C. variabilis proteins E1ZMT9 and E1Z2I7 also share sequence similarity with the crystallized N-terminus of NELF-A (25% identity, 44% similarity) and C-terminal region of NELF-C (23% identity, 39% similarity). The conservation of many residues in the hydrophobic core and heterodimer interface indicates that the NELF-AC subcomplex exists in this single cell organism. A putative Dictyostelium homolog is also found for a region of human NELF-B comprising residues 1–410 (DDB_G0284195) (Chang et al., 2012). NELF is likely present in both single and multicellular organisms. Plants may also lack NELF, indicating that they may be devoid of promoter proximal pausing or use other mechanisms to regulate gene expression at the level of elongation. Future work would benefit from understanding how NELF evolved and why some organisms either lost or lack the complex.DOI:http://dx.doi.org/10.7554/eLife.14981.005
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fig1s3: Multiple sequence alignment of the N-terminal region of NELF-C.The alignment compares full-length NELF-C from Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster, Dictyostelium discoideum, Loa loa, Lottia gigantea, and Chlorella variabilis. Residues are colored according to percent conservation with darker shades of blue representing higher conservation. Barrels above the alignment represent α-helices, arrows β-sheets and are colored according to Figure 1B. N- and C-terminal borders of solved crystal structure are indicated. Sequence alignment was done with Mafft (Katoh and Standley, 2013) followed by manual editing and rendered with JALVIEW (Waterhouse et al., 2009). Surprisingly, a part of the NELF-AC complex also exists in the single cell slime mold Dictyostelium discoideum and the green algae Chlorella variabilis. The hypothetical Dictyostelium proteins DDB_G0286295 and DDB_G0268678 share sequence similarity with the crystallized N-terminus of NELF-A (28% identity, 50% similarity) and C-terminal region of NELF-C (33% identity, 53% similarity) (Figure 1B). Similarly, the hypothetical C. variabilis proteins E1ZMT9 and E1Z2I7 also share sequence similarity with the crystallized N-terminus of NELF-A (25% identity, 44% similarity) and C-terminal region of NELF-C (23% identity, 39% similarity). The conservation of many residues in the hydrophobic core and heterodimer interface indicates that the NELF-AC subcomplex exists in this single cell organism. A putative Dictyostelium homolog is also found for a region of human NELF-B comprising residues 1–410 (DDB_G0284195) (Chang et al., 2012). NELF is likely present in both single and multicellular organisms. Plants may also lack NELF, indicating that they may be devoid of promoter proximal pausing or use other mechanisms to regulate gene expression at the level of elongation. Future work would benefit from understanding how NELF evolved and why some organisms either lost or lack the complex.DOI:http://dx.doi.org/10.7554/eLife.14981.005
Mentions: The crystallized regions of human NELF-AC share considerable homology among metazoans, particularly at residues forming the hydrophobic cores and the interface between NELF-A and NELF-C (Figure 1B). The extent of conservation is evident when human and Drosophila melanogaster are compared, which share 55% identity for NELF-A and 50% identity for NELF-C. Intriguingly, NELF-A and -C homologs are present in some worms such as the filiarial nematode Loa loa (Figure 1—figure supplements 2, 3) and single celled organisms such as the green algae Chlorella variabilis and the slime mold Dictyostelium discoideum (Figure 1—figure supplements 2, 3). Most regions outside of the crystallized NELF-AC core diverge between single celled organisms and metazoans (Figure 1—figure supplements 2, 3). Such conservation suggests that NELF may have been present in early eukaryotes and was lost in certain lineages over time.

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.